Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins

ABSTRACT

Disclosed are synthetic nanocarrier compositions, and related methods, comprising therapeutic protein APC presentable antigens and immunosuppressants that provide tolerogenic immune responses specific to therapeutic proteins.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/458,067, filed Apr. 27, 2012, pending, which claims the benefit under35 U.S.C. §119 of U.S. provisional application 61/480,946, filed Apr.29, 2011, 61/513,514, filed Jul. 29, 2011, 61/531,147, filed Sep. 6,2011, 61/531,153, filed Sep. 6, 2011, 61/531,164, filed Sep. 6, 2011,61/531,168, filed Sep. 6, 2011, 61/531,175, filed Sep. 6, 2011,61/531,180, filed Sep. 6, 2011, 61/531,194, filed Sep. 6, 2011,61/531,204, filed Sep. 6, 2011, 61/531,209, filed Sep. 6, 2011, and61/531,215, filed Sep. 6, 2011, the entire contents of each of which areincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to synthetic nanocarrier compositions withtherapeutic protein antigen-presenting cell (APC) presentable antigensand immunosuppressants, and related methods. The compositions andmethods allow for efficient uptake by APCs to shift the immune responsein favor of tolerogenic immune response development specific totherapeutic proteins. The compositions and methods provided can,therefore, be used to generate a tolerogenic immune response in asubject in which the administration of a therapeutic protein is or isexpected to result in undesired immune responses.

BACKGROUND OF THE INVENTION

Therapeutic treatments, such as protein or enzyme replacement therapies,often result in undesired immune responses to the particulartherapeutic. In such cases, cells of the immune system recognize thetherapeutic as foreign and attempt to destroy it, just as they attemptto destroy infecting organisms such as bacteria and viruses. Suchundesired immune responses may be reduced through the use ofimmunosuppressant drugs. Conventional immunosuppressant drugs, however,are broad-acting. Additionally, in order to maintain immunosuppression,immunosuppressant drug therapy is generally a life-long proposition.Unfortunately, the use of broad-acting immunosuppressants are associatedwith a risk of severe side effects, such as tumors, infections,nephrotoxicity and metabolic disorders. Accordingly, newimmunosuppressant therapies would be beneficial.

SUMMARY OF THE INVENTION

In one aspect, a composition comprising (i) a first population ofsynthetic nanocarriers that are coupled to immunosuppressants, and (ii)a second population of synthetic nanocarriers that are coupled totherapeutic protein APC presentable antigens is provided. In oneembodiment, the first population and second population are the samepopulation. In another embodiment. The first population and secondpopulation are different populations.

In yet another embodiment, the immunosuppressants comprise a statin, anmTOR inhibitor, a TGF-β signaling agent, a corticosteroid, an inhibitorof mitochondrial function, a P38 inhibitor, an NF-κβ inhibitor, anadenosine receptor agonist, a prostaglandin E2 agonist, aphosphodiesterasse 4 inhibitor, an HDAC inhibitor or a proteasomeinhibitor. In still another embodiment, the mTOR inhibitor is rapamycinor a rapamycin analog.

In some embodiments, the therapeutic protein APC presentable antigensare provided by coupling the therapeutic protein to the syntheticnanocarriers. In other embodiments, the therapeutic protein APCpresentable antigens are provided by coupling a polypeptide or peptideobtained or derived from the therapeutic protein. In a furtherembodiment, the therapeutic protein APC presentable antigens compriseMHC Class I-restricted and/or MHC Class II-restricted epitopes and/or Bcell epitopes of a therapeutic protein. In another embodiment, thetherapeutic protein APC presentable antigens comprise MHC ClassII-restricted epitopes of a therapeutic protein. In another embodiment,the therapeutic protein APC presentable antigens comprise substantiallyno B cell epitopes of a therapeutic protein. In another embodiment, thetherapeutic protein APC presentable antigens comprise MHC ClassII-restricted epitopes and substantially no B cell epitopes of atherapeutic protein.

In one embodiment, the therapeutic protein APC presentable antigenscomprise a therapeutic protein for protein replacement or proteinsupplementation therapy, or a fragment or derivative thereof. In anotherembodiment, the therapeutic protein APC presentable antigens comprisea/an infusible or injectable therapeutic protein, enzyme, enzymecofactor, hormone, blood or blood coagulation factor, cytokine,interferon, growth factor, monoclonal antibody, polyclonal antibody orprotein associated with Pompe's disease, or a fragment or derivative ofany of the foregoing. In another embodiment, the infusible or injectabletherapeutic protein comprises Tocilizumab, alpha-1 antitrypsin,Hematide, albinterferon alfa-2b, Rhucin, tesamorelin, ocrelizumab,belimumab, pegloticase, taliglucerase alfa, agalsidase alfa orvelaglucerase alfa. In another embodiment, the enzyme comprises anoxidoreductase, transferase, hydrolase, lyase, isomerase or ligase. Inanother embodiment, the enzyme comprises an enzyme for enzymereplacement therapy for a lysosomal storage disorder. In anotherembodiment, the enzyme for enzyme replacement therapy for a lysosomalstorage disorder comprises imiglucerase, a-galactosidase A (a-gal A),agalsidase beta, acid a-glucosidase (GAA), alglucosidase alfa, LUMIZYME,MYOZYME, arylsulfatase B, laronidase, ALDURAZYME, idursulfase, ELAPRASE,arylsulfatase B or NAGLAZYME. In another embodiment, the cytokinecomprises a lymphokine, interleukin, chemokine, type 1 cytokine or atype 2 cytokine. In another embodiment, the blood and blood coagulationfactor comprises Factor I, Factor II, tissue factor, Factor V, FactorVII, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XII, FactorXIII, von Willebrand factor, prekallikrein, high-molecular weightkininogen, fibronectin, antithrombin III, heparin cofactor II, proteinC, protein S, protein Z, protein Z-related protease inhibitor (ZPI),plasminogen, alpha 2-antiplasmin, tissue plasminogen activator (tPA),urokinase, plasminogen activator inhibitor-1 (PAI1), plasminogenactivator inhibitor-2 (PAI2), cancer procoagulant or epoetin alfa. Instill another embodiment, the therapeutic protein is expressed in, by oron cells of a cell-based therapy.

In one embodiment, the composition is in an amount effective to generatea tolerogenic immune response to the therapeutic protein APC presentableantigen or therapeutic protein from which the antigens are obtained orderived. In another embodiment, the composition is in an amounteffective to reduce the generation of therapeutic protein-specificantibodies when administered to a subject.

In another embodiment, the load of the immunosuppressants and/orantigens on average across the first and/or second population ofsynthetic nanocarriers is between 0.0001% and 50%. In still anotherembodiment, the load of the immunosuppressants and/or antigens onaverage across the first and/or second population of syntheticnanocarriers is between 0.1% and 10%.

In one embodiment, the synthetic nanocarriers of the first populationand/or second population comprise lipid nanoparticles, polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, buckyballs, nanowires, virus-like particles or peptide orprotein particles. In another embodiment, the synthetic nanocarriers ofthe first and/or second populations comprise lipid nanoparticles. In yetanother embodiment, the synthetic nanocarriers of the first and/orsecond populations comprise liposomes. In still another embodiment, thesynthetic nanocarriers of the first and/or second populations comprisemetallic nanoparticles. In a further embodiment, the metallicnanoparticles comprise gold nanoparticles. In yet a further embodiment,the synthetic nanocarriers of the first and/or second populationscomprise polymeric nanoparticles. In another embodiment, the polymericnanoparticles comprise polymers that are non-methoxy-terminated,pluronic polymers. In still a further embodiment, the polymericnanoparticles comprise a polyester, a polyester coupled to a polyether,polyamino acid, polycarbonate, polyacetal, polyketal, polysaccharide,polyethyloxazoline or polyethyleneimine. In another embodiment, thepolyester comprises a poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid) or polycaprolactone. In yet anotherembodiment, the polymeric nanoparticles comprise a polyester and apolyester coupled to a polyether. In another embodiment, the polyethercomprises polyethylene glycol or polypropylene glycol.

In another embodiment, the mean of a particle size distribution obtainedusing dynamic light scattering of the synthetic nanocarriers of thefirst and/or second population is a diameter greater than 100 nm. Inanother embodiment, the diameter is greater than 150 nm. In anotherembodiment, the diameter is greater than 200 nm. In another embodiment,the diameter is greater than 250 nm. In another embodiment, the diameteris greater than 300 nm.

In a further embodiment, the aspect ratio of the synthetic nanocarriersof the first population and/or second population is greater than 1:1,1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.

In yet another embodiment, the composition further comprises apharmaceutically acceptable excipient.

In another aspect, a dosage form comprising any of the compositionsprovided herein is provided.

In another aspect, a method comprising administering any of thecompositions or dosage forms provided herein to a subject that is beingadministered or will be administered a therapeutic protein is provided.In one embodiment, the method further comprises administering thetherapeutic protein to the subject. In another embodiment, thetherapeutic protein is administered prior to, concomitantly with orafter the administration of the composition or dosage form. In yetanother embodiment, one or more maintenance doses of the composition ordosage form is administered to the subject.

In yet another embodiment, the method further comprises assessing thegeneration of an undesired immune response in the subject prior toand/or after the administration of the composition or dosage form and/orthe therapeutic protein. In one embodiment, the undesired immuneresponse is the generation of therapeutic protein-specific antibodies.In another embodiment, the undesired immune response is CD4+ T cellproliferation and/or activity specific to the therapeutic protein. Inyet another embodiment, the undesired immune response is B cellproliferation and/or activity specific to the therapeutic protein.

In one embodiment, the therapeutic protein comprises a therapeuticprotein for protein replacement or protein supplementation therapy. Inanother embodiment, the therapeutic protein comprises a/an infusible orinjectable therapeutic protein, enzyme, enzyme cofactor, hormone, bloodor blood coagulation factor, cytokine, interferon, growth factor,monoclonal antibody, polyclonal antibody or protein associated withPompe's disease. In another embodiment, the infusible or injectabletherapeutic protein comprises Tocilizumab, alpha-1 antitrypsin,Hematide, albinterferon alfa-2b, Rhucin, tesamorelin, ocrelizumab,belimumab, pegloticase, taliglucerase alfa, agalsidase alfa orvelaglucerase alfa. In another embodiment, the enzyme comprises anoxidoreductase, transferase, hydrolase, lyase, isomerase or ligase. Inanother embodiment, the enzyme comprises an enzyme for enzymereplacement therapy for a lysosomal storage disorder. In anotherembodiment, the enzyme for enzyme replacement therapy for a lysosomalstorage disorder comprises imiglucerase, a-galactosidase A (a-gal A),agalsidase beta, acid a-glucosidase (GAA), alglucosidase alfa, LUMIZYME,MYOZYME, arylsulfatase B, laronidase, ALDURAZYME, idursulfase, ELAPRASE,arylsulfatase B or NAGLAZYME. In another embodiment, the cytokinecomprises a lymphokine, interleukin, chemokine, type 1 cytokine or atype 2 cytokine. In another embodiment, the blood and blood coagulationfactor comprises Factor I, Factor II, tissue factor, Factor V, FactorVII, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XII, FactorXIII, von Willebrand factor, prekallikrein, high-molecular weightkininogen, fibronectin, antithrombin III, heparin cofactor II, proteinC, protein S, protein Z, protein Z-related protease inhibitor (ZPI),plasminogen, alpha 2-antiplasmin, tissue plasminogen activator (tPA),urokinase, plasminogen activator inhibitor-1 (PAI1), plasminogenactivator inhibitor-2 (PAI2), cancer procoagulant or epoetin alfa. Instill another embodiment, the therapeutic protein is expressed in, by oron cells of a cell-based therapy.

In one embodiment, the administering of the first and/or secondpopulation of synthetic nanocarriers and/or therapeutic protein is byintravenous, intraperitoneal, transmucosal, oral, subcutaneous,pulmonary, intranasal, intradermal or intramuscular administration. Inanother embodiment, the administering of the first and/or secondpopulation of synthetic nanocarriers and/or therapeutic protein is byinhalation or intravenous, subcutaneous or transmucosal administration.

In still another aspect, a method comprising administering to a subjecta composition comprising (i) a first population of syntheticnanocarriers that are coupled to immunosuppressants, and (ii) a secondpopulation of synthetic nanocarriers that are coupled to therapeuticprotein APC presentable antigens, wherein the composition is in anamount effective to reduce the generation of an undesired immuneresponse against the therapeutic protein APC presentable antigens isprovided. In yet another aspect, a method comprising reducing thegeneration of an undesired immune response in a subject by administeringa composition comprising (i) a first population of syntheticnanocarriers that are coupled to immunosuppressants, and (ii) a secondpopulation of synthetic nanocarriers that are coupled to therapeuticprotein APC presentable antigens is provided. In a further aspect, amethod comprising administering a composition to a subject according toa protocol that was previously shown to reduce the generation of anundesired immune response to therapeutic protein APC presentableantigens in one or more test subjects; wherein the composition comprises(i) a first population of synthetic nanocarriers that are coupled toimmunosuppressants, and (ii) a second population of syntheticnanocarriers that are coupled to the therapeutic protein APC presentableantigens is provided. In one embodiment, the first population and secondpopulation are the same population. In another embodiment, the firstpopulation and second population are different populations.

In yet another embodiment, the method further comprises providing oridentifying the subject. In still another embodiment, the method furthercomprises assessing the generation of the undesired immune response inthe subject prior to and/or after the administration of the composition.In one embodiment, the undesired immune response is the generation oftherapeutic protein-specific antibodies and/or CD4+ T cell proliferationand/or activity specific to the therapeutic protein and/or B cellproliferation and/or activity specific to the therapeutic protein.

In another embodiment, the immunosuppressants comprise a statin, an mTORinhibitor, a TGF-β signaling agent, a corticosteroid, an inhibitor ofmitochondrial function, a P38 inhibitor, an NF-κβ inhibitor, anadenosine receptor agonist, a prostaglandin E2 agonist, aphosphodiesterasse 4 inhibitor, an HDAC inhibitor or a proteasomeinhibitor. In yet another embodiment, the mTOR inhibitor is rapamycin ora rapamycin analog.

In some embodiments, the therapeutic protein APC presentable antigensare provided by coupling the therapeutic protein to the syntheticnanocarriers. In other embodiments, the therapeutic protein APCpresentable antigens are provided by coupling a polypeptide or peptideobtained or derived from the therapeutic protein. In a furtherembodiment, the therapeutic protein APC presentable antigens compriseMHC Class I-restricted and/or MHC Class II-restricted epitopes and/or Bcell epitopes of a therapeutic protein. In another embodiment, thetherapeutic protein APC presentable antigens comprise MHC ClassII-restricted epitopes of a therapeutic protein. In another embodiment,the therapeutic protein APC presentable antigens comprise substantiallyno B cell epitopes of a therapeutic protein. In another embodiment, thetherapeutic protein APC presentable antigens comprise MHC ClassII-restricted epitopes and substantially no B cell epitopes of atherapeutic protein.

In one embodiment, the therapeutic protein comprises a therapeuticprotein for protein replacement or protein supplementation therapy. Inanother embodiment, the therapeutic protein comprises a/an infusible orinjectable therapeutic protein, enzyme, enzyme cofactor, hormone, bloodor blood coagulation factor, cytokine, interferon, growth factor,monoclonal antibody, polyclonal antibody or protein associated withPompe's disease. In another embodiment, the infusible or injectabletherapeutic protein comprises Tocilizumab, alpha-1 antitrypsin,Hematide, albinterferon alfa-2b, Rhucin, tesamorelin, ocrelizumab,belimumab, pegloticase, taliglucerase alfa, agalsidase alfa orvelaglucerase alfa. In another embodiment, the enzyme comprises anoxidoreductase, transferase, hydrolase, lyase, isomerase or ligase. Inanother embodiment, the enzyme comprises an enzyme for enzymereplacement therapy for a lysosomal storage disorder. In anotherembodiment, the enzyme for enzyme replacement therapy for a lysosomalstorage disorder comprises imiglucerase, a-galactosidase A (a-gal A),agalsidase beta, acid a-glucosidase (GAA), alglucosidase alfa, LUMIZYME,MYOZYME, arylsulfatase B, laronidase, ALDURAZYME, idursulfase, ELAPRASE,arylsulfatase B or NAGLAZYME. In another embodiment, the cytokinecomprises a lymphokine, interleukin, chemokine, type 1 cytokine or atype 2 cytokine. In another embodiment, the blood and blood coagulationfactor comprises Factor I, Factor II, tissue factor, Factor V, FactorVII, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XII, FactorXIII, von Willebrand factor, prekallikrein, high-molecular weightkininogen, fibronectin, antithrombin III, heparin cofactor II, proteinC, protein S, protein Z, protein Z-related protease inhibitor (ZPI),plasminogen, alpha 2-antiplasmin, tissue plasminogen activator (tPA),urokinase, plasminogen activator inhibitor-1 (PAI1), plasminogenactivator inhibitor-2 (PAI2), cancer procoagulant or epoetin alfa. Instill another embodiment, the therapeutic protein is expressed in, by oron cells of a cell-based therapy.

In one embodiment, the composition reduces is in an amount effective toreduce the generation of therapeutic protein-specific antibodies and/orCD4+ T cell proliferation and/or activity specific to the therapeuticprotein and/or B cell proliferation and/or activity specific to thetherapeutic protein.

In another embodiment, the load of the immunosuppressants and/orantigens on average across the first and/or second population ofsynthetic nanocarriers is between 0.0001% and 50%. In still anotherembodiment, the load of the immunosuppressants and/or antigens onaverage across the first and/or second population of syntheticnanocarriers is between 0.1% and 10%.

In one embodiment, the synthetic nanocarriers of the first populationand/or second population comprise lipid nanoparticles, polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, buckyballs, nanowires, virus-like particles or peptide orprotein particles. In another embodiment, the synthetic nanocarriers ofthe first and/or second populations comprise lipid nanoparticles. In yetanother embodiment, the synthetic nanocarriers of the first and/orsecond populations comprise liposomes. In still another embodiment, thesynthetic nanocarriers of the first and/or second populations comprisemetallic nanoparticles. In a further embodiment, the metallicnanoparticles comprise gold nanoparticles. In yet a further embodiment,the synthetic nanocarriers of the first and/or second populationscomprise polymeric nanoparticles. In another embodiment, the polymericnanoparticles comprise polymers that are non-methoxy-terminated,pluronic polymers. In still a further embodiment, the polymericnanoparticles comprise a polyester, a polyester coupled to a polyether,polyamino acid, polycarbonate, polyacetal, polyketal, polysaccharide,polyethyloxazoline or polyethyleneimine. In another embodiment, thepolyester comprises a poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid) or polycaprolactone. In yet anotherembodiment, the polymeric nanoparticles comprise a polyester and apolyester coupled to a polyether. In still another embodiment, thepolyether comprises polyethylene glycol or polypropylene glycol.

In another embodiment, the mean of a particle size distribution obtainedusing dynamic light scattering of the synthetic nanocarriers of thefirst and/or second population is a diameter greater than 100 nm. Inanother embodiment, the diameter is greater than 150 nm. In anotherembodiment, the diameter is greater than 200 nm. In another embodiment,the diameter is greater than 250 nm. In another embodiment, the diameteris greater than 300 nm.

In a further embodiment, the aspect ratio of the synthetic nanocarriersof the first population and/or second population is greater than 1:1,1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.

In still a further embodiment, the composition further comprises apharmaceutically acceptable excipient.

In yet a further embodiment, the method further comprises administeringthe therapeutic protein to the subject. In one embodiment, thetherapeutic protein is administered prior to, concomitantly with orafter the administration of the composition.

In another embodiment, one or more maintenance doses of the compositionis administered to the subject.

In still another embodiment, the method further comprises assessing thegeneration of an undesired immune response in the subject prior toand/or after the administration of the composition and/or therapeuticprotein. In another embodiment, the undesired immune response is thegeneration of therapeutic protein-specific antibodies and/or CD4+ T cellproliferation and/or activity specific to the therapeutic protein and/orB cell proliferation and/or activity specific to the therapeuticprotein.

In one embodiment, the administering of the first and/or secondpopulation of synthetic nanocarriers and/or therapeutic protein is byintravenous, intraperitoneal, transmucosal, oral, subcutaneous,pulmonary, intranasal, intradermal or intramuscular administration. Inanother embodiment, the administering of the first and/or secondpopulation of synthetic nanocarriers and/or therapeutic protein is byinhalation or intravenous, subcutaneous or transmucosal administration.

In a further aspect, a method comprising (i) producing a firstpopulation of synthetic nanocarriers that are coupled toimmunosuppressants, and (ii) producing a second population of syntheticnanocarriers that are coupled to therapeutic protein APC presentableantigens is provided. In one embodiment, the first population and secondpopulation are the same population. In another embodiment, the firstpopulation and second population are different populations. In yetanother embodiment, the first and second populations of syntheticnanocarriers that are produced are as defined as provided anywhereherein.

In one embodiment, the method further comprises producing a dosage formof a composition comprising the first and second populations ofsynthetic nanocarriers produced. In still another embodiment, the methodfurther comprises making a composition comprising the first populationand second population of synthetic nanocarriers or the dosage formavailable to a subject for administration. In a further embodiment, themethod further comprises assessing the reduction of an undesired immuneresponse with a composition comprising the first population and secondpopulation of synthetic nanocarriers. In one embodiment, the undesiredimmune response is the generation of therapeutic protein-specificantibodies and/or CD4+ T cell proliferation and/or activity specific tothe therapeutic protein and/or B cell proliferation and/or activityspecific to the therapeutic protein.

In yet a further aspect, a process for producing a composition or dosageform comprising (i) a first population of synthetic nanocarriers thatare coupled to immunosuppressants, and (ii) a second population ofsynthetic nanocarriers that are coupled to therapeutic protein APCpresentable antigens, the process comprising the steps as defined in anyof the methods provided herein is provided.

In another aspect, a composition or dosage form obtainable by any of themethods or processes provided herein is provided.

In yet another aspect, any of the compositions or dosage forms providedherein may be for use in therapy or prophylaxis.

In still another aspect, any of the compositions or dosage formsprovided herein may be for use in a method of inducing a tolerogenicimmune response to therapeutic protein antigens, cell-based therapy,protein replacement therapy, protein supplementation therapy or in anyof the methods provided herein.

In a further aspect, use of any of the compositions or dosage formsprovided herein for the manufacture of a medicament for use in a methodof inducing a tolerogenic immune response to therapeutic proteinantigens, cell-based therapy, protein replacement therapy, proteinsupplementation therapy or in any of the methods provided herein isprovided.

In still another aspect, a dosage form comprising any of thecompositions provided herein is provided.

In some embodiments of any of the compositions and methods provided, thecompositions provided herein are administered prophylactically, early inthe immune response (e.g., during an IgM phase) and/or prior toestablishment of a mature memory response and/or an IgG antibodyresponse. In embodiments of any of the compositions and methods providedherein, the compositions and methods may reduce the frequency ofeffector T cells reacting to antigen and/or their capacity to producepro-inflammatory cytokines. In other embodiments of any of thecompositions and methods provided herein, the compositions and methodsincrease the frequency of suppressive regulatory T or B cells thatreduce the incidence of undesired therapeutic-protein immune responses.

In an embodiment of any of the compositions and methods provided herein,antigens that are proteins that comprise the aforementioned epitopes canbe coupled to the synthetic nanocarriers. In another embodiment,polypeptides or peptides that comprise the aforementioned epitopes butadditional amino acids that flank one or both ends of the epitope(s) canbe coupled to the synthetic nanocarriers. In another embodiment, theepitopes themselves are coupled to the synthetic nanocarriers.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows results from a flow cytometric analysis of Treg.

FIG. 2 shows an effect on the number of antigen-specific effector Tcells with synthetic nanocarriers of the invention comprisingimmunosuppressant (rapamycin or simvastatin) (after a single injection).

FIG. 3 shows a decrease in the number of popliteal lymph node cells withsynthetic nanocarriers of the invention comprising immunosuppressant(rapamycin or simvastatin) (after multiple injections).

FIG. 4 demonstrates the reduction of anti-OVA IgG antibodies withsynthetic nanocarriers that comprise the immunosuppressant rapamycin andova antigen.

FIG. 5 demonstrates in the control and passive groups the reduction ofanti-OVA IgG antibodies with synthetic nanocarriers that comprise theimmunosuppressant rapamycin and OVA antigen.

FIG. 6 shows a reduction in antigen-specific IgG levels with theadministration of synthetic nanocarriers comprising ova peptide and theimmunosuppressant rapamycin.

FIG. 7 demonstrates a reduction in the number of antigen-specific Bcells with synthetic nanocarriers comprising ova peptide and theimmunosuppressant rapamycin.

FIG. 8 demonstrates a reduction in the number of CD4+ T cells in lavagesamples from asthma model animal subjects treated with syntheticnanocarriers comprising ova peptide and immunosuppressant.

FIG. 9 demonstrates a reduction in the percentage of dividing CD4+ Tcells as a result of treatment with synthetic nanocarriers comprisingova peptide and the immunosuppressant rapamycin in asthma model animalsubjects.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified materials or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting of the use of alternativeterminology to describe the present invention.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyfor all purposes.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a polymer”includes a mixture of two or more such molecules or a mixture ofdiffering molecular weights of a single polymer species, reference to “asynthetic nanocarrier” includes a mixture of two or more such syntheticnanocarriers or a plurality of such synthetic nanocarriers, reference to“a DNA molecule” includes a mixture of two or more such DNA molecules ora plurality of such DNA molecules, reference to “an immunosuppressant”includes a mixture of two or more such materials or a plurality ofimmunosuppressant molecules, and the like.

As used herein, the term “comprise” or variations thereof such as“comprises” or “comprising” are to be read to indicate the inclusion ofany recited integer (e.g. a feature, element, characteristic, property,method/process step or limitation) or group of integers (e.g. features,element, characteristics, properties, method/process steps orlimitations) but not the exclusion of any other integer or group ofintegers. Thus, as used herein, the term “comprising” is inclusive anddoes not exclude additional, unrecited integers or method/process steps.

In embodiments of any of the compositions and methods provided herein,“comprising” may be replaced with “consisting essentially of” or“consisting of”. The phrase “consisting essentially of” is used hereinto require the specified integer(s) or steps as well as those which donot materially affect the character or function of the claimedinvention. As used herein, the term “consisting” is used to indicate thepresence of the recited integer (e.g. a feature, element,characteristic, property, method/process step or limitation) or group ofintegers (e.g. features, element, characteristics, properties,method/process steps or limitations) alone.

A. INTRODUCTION

As previously mentioned, current conventional immunosuppressants arebroad acting and generally result in an overall systemic downregulationof the immune system. The compositions and methods provided herein allowfor more targeted immune effects by, for example, allowing for thetargeted delivery to immune cells of interest. Thus, the compositionsand methods can achieve immune suppression in a more directed manner. Ithas been found that delivering immunosuppressants and antigenscomprising MHC Class II-restricted epitopes can effectively reduce theproduction of antigen-specific antibodies as well as reduce thegeneration of antigen-specific CD4+ T cells and B cells. As such immuneresponses can be beneficial in countering undesired immune responsesthat are generated during therapeutic treatment with therapeuticproteins, the invention is useful for promoting tolerogenic immuneresponses in subjects who have received, are receiving or will receive atherapeutic protein against which undesired immune responses aregenerated or are expected to be generated. The present invention, insome embodiments, prevents or suppresses undesired immune responses thatmay neutralize the beneficial effect of certain therapeutic treatments.

The inventors have unexpectedly and surprisingly discovered that theproblems and limitations noted above can be overcome by practicing theinvention disclosed herein. In particular, the inventors haveunexpectedly discovered that it is possible to provide syntheticnanocarrier compositions, and related methods, that induce a tolerogenicimmune response to therapeutic proteins. The compositions describedherein include compositions that comprise (i) a first population ofsynthetic nanocarriers that are coupled to immunosuppressants, and (ii)a second population of synthetic nanocarriers that are coupled totherapeutic protein APC presentable antigens.

In another aspect, dosage forms of any of the compositions herein areprovided. Such dosage forms can be administered to a subject, such as asubject in need thereof (e.g., in need of therapeutic protein-specifictolerogenic immune responses). In one embodiment, the subject is one whohas been, is being or will be treated with a therapeutic protein againstwhich undesired immune responses are generated or are expected to begenerated.

In another aspect, any of the compositions provided herein isadministered to a subject. The composition may be administered in anamount effective to reduce the generation of an undesired immuneresponse against one or more therapeutic proteins. In one embodiment, acomposition is administered to a subject according to a protocol thatwas previously shown to reduce the generation of an undesired immuneresponse to therapeutic proteins in one or more subjects. The undesiredimmune response may be the generation of therapeutic protein-specificantibodies, CD4+ T cell proliferation and/or activity or B cellproliferation and/or activity or combinations thereof. The undesiredimmune responses may also be the generation of other immune responsesdownstream from the foregoing. In embodiments, the amounts effective, orprotocol, generates, or has been shown to generate desired immuneresponses. Such immune responses include the reduction of any of, orcombinations of, the foregoing. Such immune responses include anytolerogenic immune responses, such as those described herein.

The compositions may be administered to a subject prior to,concomitantly with or after the administration of a therapeutic proteinto the subject. In some embodiments, the synthetic nanocarrierscompositions are administered prior to the establishment of a maturememory response in the subject. The methods provided, in someembodiments, may further comprise administering the therapeutic protein.In embodiments, the compositions provided may also be administered asone or more maintenance doses to a subject that has received, isreceiving or will receive a therapeutic protein. In such embodiments,the compositions provided are administered such that the generation ofan undesired immune response is reduced for a certain length of time.Examples of such lengths of time are provided elsewhere herein.

In yet another aspect, a method of (i) producing a first population ofsynthetic nanocarriers that are coupled to immunosuppressants, and (ii)producing a second population of synthetic nanocarriers that are coupledto therapeutic protein APC presentable antigens is provided. In oneembodiment, the method further comprises producing a dosage formcomprising the first and second populations of synthetic nanocarriers.

The invention will now be described in more detail below.

B. DEFINITIONS

“Administering” or “administration” means providing a material to asubject in a manner that is pharmacologically useful.

“Amount effective” in the context of a composition or dosage form foradministration to a subject refers to an amount of the composition ordosage form that produces one or more desired immune responses in thesubject, for example, the generation of a tolerogenic immune response(e.g, a reduction in the proliferation, activation, induction,recruitment of antigen-specific CD4+ T cells or antigen-specific B cellsor a reduction in the production of antigen-specific antibodies).Therefore, in some embodiments, an amount effective is any amount of acomposition provided herein that produces one or more of these desiredimmune responses. This amount can be for in vitro or in vivo purposes.For in vivo purposes, the amount can be one that a clinician wouldbelieve may have a clinical benefit for a subject in need ofantigen-specific tolerization.

Amounts effective can involve only reducing the level of an undesiredimmune response, although in some embodiments, it involves preventing anundesired immune response altogether. Amounts effective can also involvedelaying the occurrence of an undesired immune response. An amount thatis effective can also be an amount of a composition provided herein thatproduces a desired therapeutic endpoint or a desired therapeutic result.Amounts effective, preferably, result in a tolerogenic immune responsein a subject to an antigen. The achievement of any of the foregoing canbe monitored by routine methods.

In some embodiments of any of the compositions and methods provided, theamount effective is one in which the desired immune response persists inthe subject for at least 1 week, at least 2 weeks, at least 1 month, atleast 2 months, at least 3 months, at least 4 months, at least 5 months,at least 6 months, at least 9 months, at least 1 year, at least 2 years,at least 5 years, or longer. In other embodiments of any of thecompositions and methods provided, the amount effective is one whichproduces a measurable desired immune response, for example, a measurabledecrease in an immune response (e.g., to a specific antigen), for atleast 1 week, at least 2 weeks, at least 1 month, at least 2 months, atleast 3 months, at least 4 months, at least 5 months, at least 6 months,at least 9 months, at least 1 year, at least 2 years, at least 5 years,or longer.

Amounts effective will depend, of course, on the particular subjectbeing treated; the severity of a condition, disease or disorder; theindividual patient parameters including age, physical condition, sizeand weight; the duration of the treatment; the nature of concurrenttherapy (if any); the specific route of administration and like factorswithin the knowledge and expertise of the health practitioner. Thesefactors are well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. It is generallypreferred that a maximum dose be used, that is, the highest safe doseaccording to sound medical judgment. It will be understood by those ofordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reason.

In general, doses of the immunosuppressants and/or antigens in thecompositions of the invention can range from about 10 μg/kg to about100,000 μg/kg. In some embodiments, the doses can range from about 0.1mg/kg to about 100 mg/kg. In still other embodiments, the doses canrange from about 0.1 mg/kg to about 25 mg/kg, about 25 mg/kg to about 50mg/kg, about 50 mg/kg to about 75 mg/kg or about 75 mg/kg to about 100mg/kg. Alternatively, the dose can be administered based on the numberof synthetic nanocarriers that provide the desired amount ofimmunosuppressants and/or antigens. For example, useful doses includegreater than 10⁶, 10⁷, 10⁸, 10⁹ or 10¹⁰ synthetic nanocarriers per dose.Other examples of useful doses include from about 1×10⁶ to about 1×10¹⁰,about 1×10⁷ to about 1×10⁹ or about 1×10⁸ to about 1×10⁹ syntheticnanocarriers per dose.

“Antigen” means a B cell antigen or T cell antigen. “Type(s) ofantigens” means molecules that share the same, or substantially thesame, antigenic characteristics. In some embodiments, antigens may beproteins, polypeptides, peptides, lipoproteins, glycolipids,polynucleotides, polysaccharides or are contained or expressed in cells.In some embodiments, such as when the antigens are not well defined orcharacterized, the antigens may be contained within a cell or tissuepreparation, cell debris, cell exosomes, conditioned media, etc. Anantigen can be combined with the synthetic nanocarriers in the same formas what a subject is exposed to that causes an undesired immune responsebut may also be a fragment or derivative thereof. When a fragment orderivative, however, a desired immune response to the form encounteredby such a subject is the preferable result with the compositions andmethods provided.

“Antigen-specific” refers to any immune response that results from thepresence of the antigen, or portion thereof, or that generates moleculesthat specifically recognize or bind the antigen. For example, where theimmune response is antigen-specific antibody production, antibodies areproduced that specifically bind the antigen. As another example, wherethe immune response is antigen-specific B cell or CD4+ T cellproliferation and/or activity, the proliferation and/or activity resultsfrom recognition of the antigen, or portion thereof, alone or in complexwith MHC molecules, B cells, etc.

“Assessing an immune response” refers to any measurement ordetermination of the level, presence or absence, reduction, increase in,etc. of an immune response in vitro or in vivo. Such measurements ordeterminations may be performed on one or more samples obtained from asubject. Such assessing can be performed with any of the methodsprovided herein or otherwise known in the art.

An “at risk” subject is one in which a health practitioner believes hasa chance of having a disease, disorder or condition as provided hereinor is one a health practitioner believes has a chance of experiencing anundesired immune response as provided herein.

“Average”, as used herein, refers to the arithmetic mean unlessotherwise noted.

“B cell antigen” means any antigen that is or recognized by and triggersan immune response in a B cell (e.g., an antigen that is specificallyrecognized by a B cell or a receptor thereon). In some embodiments, anantigen that is a T cell antigen is also a B cell antigen. In otherembodiments, the T cell antigen is not also a B cell antigen. B cellantigens include, but are not limited to proteins, peptides, etc. Insome embodiments, the B cell antigen comprises a non-protein antigen(i.e., not a protein or peptide antigen).

“Concomitantly” means administering two or more substances to a subjectin a manner that is correlated in time, preferably sufficientlycorrelated in time so as to provide a modulation in an immune response.In embodiments, concomitant administration may occur throughadministration of two or more substances in the same dosage form. Inother embodiments, concomitant administration may encompassadministration of two or more substances in different dosage forms, butwithin a specified period of time, preferably within 1 month, morepreferably within 1 week, still more preferably within 1 day, and evenmore preferably within 1 hour.

“Couple” or “Coupled” or “Couples” (and the like) means to chemicallyassociate one entity (for example a moiety) with another. In someembodiments, the coupling is covalent, meaning that the coupling occursin the context of the presence of a covalent bond between the twoentities. In non-covalent embodiments, the non-covalent coupling ismediated by non-covalent interactions including but not limited tocharge interactions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. In embodiments,encapsulation is a form of coupling.

“Derived” means prepared from a material or information related to amaterial but is not “obtained” from the material. Such materials may besubstantially modified or processed forms of materials taken directlyfrom a biological material. Such materials also include materialsproduced from information related to a biological material.

“Dosage form” means a pharmacologically and/or immunologically activematerial in a medium, carrier, vehicle, or device suitable foradministration to a subject.

“Encapsulate” means to enclose at least a portion of a substance withina synthetic nanocarrier. In some embodiments, a substance is enclosedcompletely within a synthetic nanocarrier. In other embodiments, most orall of a substance that is encapsulated is not exposed to the localenvironment external to the synthetic nanocarrier. In other embodiments,no more than 50%, 40%, 30%, 20%, 10% or 5% (weight/weight) is exposed tothe local environment. Encapsulation is distinct from absorption, whichplaces most or all of a substance on a surface of a syntheticnanocarrier, and leaves the substance exposed to the local environmentexternal to the synthetic nanocarrier.

“Epitope”, also known as an antigenic determinant, is the part of anantigen that is recognized by the immune system, specifically by, forexample, antibodies, B cells, or T cells. As used herein, “MHC ClassI-restricted epitopes” are epitopes that are presented to immune cellsby MHC class I molecules found on nucleated cells. “MHC ClassII-restricted epitopes” are epitopes that are presented to immune cellsby MHC class II molecules found on antigen-presenting cells (APCs), forexample, on professional antigen-presenting immune cells, such as onmacrophages, B cells, and dendritic cells, or on non-hematopoieticcells, such as hepatocytes. “B cell epitopes” are molecular structuresthat are recognized by antibodies or B cells. In some embodiments, theepitope itself is an antigen.

A number of epitopes are known to those of skill in the art, andexemplary epitopes suitable according to some aspects of this inventioninclude, but are not limited to those listed in the Immune EpitopeDatabase (www.immuneepitope.org, Vita R, Zarebski L, Greenbaum J A,Emami H, Hoof I, Salimi N, Damle R, Sette A, Peters B. The immuneepitope database 2.0. Nucleic Acids Res. 2010 January; 38 (Databaseissue):D854-62; the entire contents of which as well as all databaseentries of IEDB version 2.4, August 2011, and particularly all epitopesdisclosed therein, are incorporated herein by reference). Epitopes canalso be identified with publicly available algorithms, for example, thealgorithms described in Wang P, Sidney J, Kim Y, Sette A, Lund O,Nielsen M, Peters B. 2010. peptide binding predictions for HLA DR, DPand DQ molecules. BMC Bioinformatics 2010, 11:568; Wang P, Sidney J, DowC, Mothé B, Sette A, Peters B. 2008. A systematic assessment of MHCclass II peptide binding predictions and evaluation of a consensusapproach. PLoS Comput Biol. 4 (4):e1000048; Nielsen M, Lund O. 2009.NN-align. An artificial neural network-based alignment algorithm for MHCclass II peptide binding prediction. BMC Bioinformatics. 10:296; NielsenM, Lundegaard C, Lund O. 2007. Prediction of MHC class II bindingaffinity using SMM-align, a novel stabilization matrix alignment method.BMC Bioinformatics. 8:238; Bui H H, Sidney J, Peters B, Sathiamurthy M,Sinichi A, Purton K A, Mothé B R, Chisari F V, Watkins D I, Sette A.2005. Immunogenetics. 57:304-314; Sturniolo T, Bono E, Ding J,Raddrizzani L, Tuereci O, Sahin U, Braxenthaler M, Gallazzi F, Protti MP, Sinigaglia F, Hammer J. 1999. Generation of tissue-specific andpromiscuous HLA ligand databases using DNA microarrays and virtual HLAclass II matrices. Nat Biotechnol. 17(6):555-561; Nielsen M, LundegaardC, Worning P, Lauemoller S L, Lamberth K, Buus S, Brunak S, Lund O.2003. Reliable prediction of T-cell epitopes using neural networks withnovel sequence representations. Protein Sci 12:1007-1017; Bui H H,Sidney J, Peters B, Sathiamurthy M, Sinichi A, Purton K A, Mothe B R,Chisari F V, Watkins D I, Sette A. 2005. Automated generation andevaluation of specific MHC binding predictive tools: ARB matrixapplications. Immunogenetics 57:304-314; Peters B, Sette A. 2005.Generating quantitative models describing the sequence specificity ofbiological processes with the stabilized matrix method. BMCBioinformatics 6:132; Chou P Y, Fasman G D. 1978. Prediction of thesecondary structure of proteins from their amino acid sequence. AdvEnzymol Relat Areas Mol Biol 47:45-148; Emini E A, Hughes J V, Perlow DS, Boger J. 1985. Induction of hepatitis A virus-neutralizing antibodyby a virus-specific synthetic peptide. J Virol 55:836-839; Karplus P A,Schulz G E. 1985. Prediction of chain flexibility in proteins.Naturwissenschaften 72:212-213; Kolaskar A S, Tongaonkar P C. 1990. Asemi-empirical method for prediction of antigenic determinants onprotein antigens. FEBS Lett 276:172-174; Parker J M, Guo D, Hodges R S.1986. New hydrophilicity scale derived from high-performance liquidchromatography peptide retention data: correlation of predicted surfaceresidues with antigenicity and X-ray-derived accessible sites.Biochemistry 25:5425-5432; Larsen J E, Lund O, Nielsen M. 2006. Improvedmethod for predicting linear B-cell epitopes. Immunome Res 2:2;Ponomarenko J V, Bourne P E. 2007. Antibody-protein interactions:benchmark datasets and prediction tools evaluation. BMC Struct Biol7:64; Haste Andersen P, Nielsen M, Lund O. 2006. Prediction of residuesin discontinuous B-cell epitopes using protein 3D structures. ProteinSci 15:2558-2567; Ponomarenko J V, Bui H, Li W, Fusseder N, Bourne P E,Sette A, Peters B. 2008. ElliPro: a new structure-based tool for theprediction of antibody epitopes. BMC Bioinformatics 9:514; Nielsen M,Lundegaard C, Blicher T, Peters B, Sette A, Justesen S, Buus S, and LundO. 2008. PLoS Comput Biol. 4 (7)e1000107. Quantitative predictions ofpeptide binding to any HLA-DR molecule of known sequence: NetMHCIIpan;the entire contents of each of which are incorporated herein byreference for disclosure of methods and algorithms for theidentification of epitopes.

“Generating” means causing an action, such as an immune response (e.g.,a tolerogenic immune response) to occur, either directly oneself orindirectly, such as, but not limited to, an unrelated third party thattakes an action through reliance on one's words or deeds.

“Identifying” is any action or set of actions that allows a clinician torecognize a subject as one who may benefit from the methods andcompositions provided herein. Preferably, the identified subject is onewho is in need of a tolerogenic immune response as provided herein. Theaction or set of actions may be either directly oneself or indirectly,such as, but not limited to, an unrelated third party that takes anaction through reliance on one's words or deeds.

“Immunosuppressant” means a compound that causes an APC to have animmunosuppressive (e.g., tolerogenic effect). An immunosuppressiveeffect generally refers to the production or expression of cytokines orother factors by the APC that reduces, inhibits or prevents an undesiredimmune response or that promotes a desired immune response. When the APCresults in an immunosuppressive effect on immune cells that recognize anantigen presented by the APC, the immunosuppressive effect is said to bespecific to the presented antigen. Such effect is also referred toherein as a tolerogenic effect. Without being bound by any particulartheory, it is thought that the immunosuppressive or tolerogenic effectis a result of the immunosuppressant being delivered to the APC,preferably in the presence of an antigen (e.g., an administered antigenor one that is already present in vivo). Accordingly, theimmunosuppressant includes compounds that provide a tolerogenic immuneresponse to an antigen that may or may not be provided in the samecomposition or a different composition. In one embodiment, theimmunosuppressant is one that causes an APC to promote a regulatoryphenotype in one or more immune effector cells. For example, theregulatory phenotype may be characterized by the inhibition of theproduction, induction, stimulation or recruitment of antigen-specificCD4+ T cells or B cells, the inhibition of the production ofantigen-specific antibodies, the production, induction, stimulation orrecruitment of Treg cells (e.g., CD4+CD25highFoxP3+ Treg cells), etc.This may be the result of the conversion of CD4+ T cells or B cells to aregulatory phenotype. This may also be the result of induction of FoxP3in other immune cells, such as CD8+ T cells, macrophages and iNKT cells.In one embodiment, the immunosuppressant is one that affects theresponse of the APC after it processes an antigen. In anotherembodiment, the immunosuppressant is not one that interferes with theprocessing of the antigen. In a further embodiment, theimmunosuppressant is not an apoptotic-signaling molecule. In anotherembodiment, the immunosuppressant is not a phospholipid.

Immunosuppressants include, but are not limited to, statins; mTORinhibitors, such as rapamycin or a rapamycin analog; TGF-β signalingagents; TGF-β receptor agonists; histone deacetylase inhibitors, such asTrichostatin A; corticosteroids; inhibitors of mitochondrial function,such as rotenone; P38 inhibitors; NF-κβ inhibitors, such as 6Bio,Dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists;prostaglandin E2 agonists (PGE2), such as Misoprostol; phosphodiesteraseinhibitors, such as phosphodiesterase 4 inhibitor (PDE4), such asRolipram; proteasome inhibitors; kinase inhibitors; G-protein coupledreceptor agonists; G-protein coupled receptor antagonists;glucocorticoids; retinoids; cytokine inhibitors; cytokine receptorinhibitors; cytokine receptor activators; peroxisomeproliferator-activated receptor antagonists; peroxisomeproliferator-activated receptor agonists; histone deacetylaseinhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3 KBinhibitors, such as TGX-221; autophagy inhibitors, such as3-Methyladenine; aryl hydrocarbon receptor inhibitors; proteasomeinhibitor I (PSI); and oxidized ATPs, such as P2X receptor blockers.Immunosuppressants also include IDO, vitamin D3, cyclosporins, such ascyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol,azathiopurine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG),FK506, sanglifehrin A, salmeterol, mycophenolate mofetil (MMF), aspirinand other COX inhibitors, niflumic acid, estriol and triptolide. Inembodiments, the immunosuppressant may comprise any of the agentsprovided herein.

The immunosuppressant can be a compound that directly provides theimmunosuppressive (e.g., tolerogenic) effect on APCs or it can be acompound that provides the immunosuppressive (e.g., tolerogenic) effectindirectly (i.e., after being processed in some way afteradministration). Immunosuppressants, therefore, include prodrug forms ofany of the compounds provided herein.

Immunosuppressants also include nucleic acids that encode the peptides,polypeptides or proteins provided herein that result in animmunosuppressive (e.g., tolerogenic) immune response. In embodiments,therefore, the immunosuppressant is a nucleic acid that encodes apeptide, polypeptide or protein that results in an immunosuppressive(e.g., tolerogenic) immune response, and it is the nucleic acid that iscoupled to the synthetic nanocarrier.

The nucleic acid may be DNA or RNA, such as mRNA. In embodiments, theinventive compositions comprise a complement, such as a full-lengthcomplement, or a degenerate (due to degeneracy of the genetic code) ofany of the nucleic acids provided herein. In embodiments, the nucleicacid is an expression vector that can be transcribed when transfectedinto a cell line. In embodiments, the expression vector may comprise aplasmid, retrovirus, or an adenovirus amongst others. Nucleic acids canbe isolated or synthesized using standard molecular biology approaches,for example by using a polymerase chain reaction to produce a nucleicacid fragment, which is then purified and cloned into an expressionvector. Additional techniques useful in the practice of this inventionmay be found in Current Protocols in Molecular Biology 2007 by JohnWiley and Sons, Inc.; Molecular Cloning: A Laboratory Manual (ThirdEdition) Joseph Sambrook, Peter MacCallum Cancer Institute, Melbourne,Australia; David Russell, University of Texas Southwestern MedicalCenter, Dallas, Cold Spring Harbor.

In embodiments, the immunosuppressants provided herein are coupled tosynthetic nanocarriers. In preferable embodiments, the immunosuppressantis an element that is in addition to the material that makes up thestructure of the synthetic nanocarrier. For example, in one embodiment,where the synthetic nanocarrier is made up of one or more polymers, theimmunosuppressant is a compound that is in addition and coupled to theone or more polymers. As another example, in one embodiment, where thesynthetic nanocarrier is made up of one or more lipids, theimmunosuppressant is again in addition and coupled to the one or morelipids. In embodiments, such as where the material of the syntheticnanocarrier also results in an immunosuppressive (e.g., tolerogenic)effect, the immunosuppressant is an element present in addition to thematerial of the synthetic nanocarrier that results in animmunosuppressive (e.g., tolerogenic) effect.

Other exemplary immunosuppressants include, but are not limited, smallmolecule drugs, natural products, antibodies (e.g., antibodies againstCD20, CD3, CD4), biologics-based drugs, carbohydrate-based drugs,nanoparticles, liposomes, RNAi, antisense nucleic acids, aptamers,methotrexate, NSAIDs; fingolimod; natalizumab; alemtuzumab; anti-CD3;tacrolimus (FK506), etc. Further immunosuppressants, are known to thoseof skill in the art, and the invention is not limited in this respect.

“Load” of the immunosuppressant or therapeutic protein APC presentableantigen is the amount of the immunosuppressant or therapeutic proteinAPC presentable antigen coupled to a synthetic nanocarrier based on thetotal weight of materials in an entire synthetic nanocarrier(weight/weight). Generally, the load is calculated as an average acrossa population of synthetic nanocarriers. In one embodiment, the load ofthe immunosuppressant on average across the first population ofsynthetic nanocarriers is between 0.0001% and 50%. In anotherembodiment, the load of the therapeutic protein APC presentable antigenon average across the first and/or second population of syntheticnanocarriers is between 0.0001% and 50%. In yet another embodiment, theload of the immunosuppressant and/or therapeutic protein APC presentableantigen is between 0.01% and 20%. In a further embodiment, the load ofthe immunosuppressant and/or therapeutic protein APC presentable antigenis between 0.1% and 10%. In still a further embodiment, the load of theimmunosuppressant and/or therapeutic protein APC presentable antigen isbetween 1% and 10%. In yet another embodiment, the load of theimmunosuppressant and/or the therapeutic protein APC presentable antigenis at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, atleast 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least6%, at least at least 7%, at least 8%, at least 9%, at least 10%, atleast 11%, at least 12%, at least 13%, at least 14%, at least 15%, atleast 16%, at least 17%, at least 18%, at least 19% or at least 20% onaverage across the population of synthetic nanocarriers. In yet afurther embodiment, the load of the immunosuppressant and/or thetherapeutic protein APC presentable antigen is 0.1%, 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% on average across thepopulation of synthetic nanocarriers. In some embodiments of the aboveembodiments, the load of the immunosuppressant and/or the therapeuticprotein APC presentable antigen is no more than 25% on average across apopulation of synthetic nanocarriers. In embodiments, the load iscalculated as described in the Examples.

In embodiments of any of the compositions and methods provided, the loadis calculated as follows: Approximately 3 mg of synthetic nanocarriersare collected and centrifuged to separate supernatant from syntheticnanocarrier pellet. Acetonitrile is added to the pellet, and the sampleis sonicated and centrifuged to remove any insoluble material. Thesupernatant and pellet are injected on RP-HPLC and absorbance is read at278 nm. The μg found in the pellet is used to calculate % entrapped(load), μg in supernatant and pellet are used to calculate total μgrecovered.

“Maintenance dose” refers to a dose that is administered to a subject,after an initial dose has resulted in an immunosuppressive (e.g.,tolerogenic) response in a subject, to sustain a desiredimmunosuppressive (e.g., tolerogenic) response. A maintenance dose, forexample, can be one that maintains the tolerogenic effect achieved afterthe initial dose, prevents an undesired immune response in the subject,or prevents the subject becoming a subject at risk of experiencing anundesired immune response, including an undesired level of an immuneresponse. In some embodiments, the maintenance dose is one that issufficient to sustain an appropriate level of a desired immune response.

“Maximum dimension of a synthetic nanocarrier” means the largestdimension of a nanocarrier measured along any axis of the syntheticnanocarrier. “Minimum dimension of a synthetic nanocarrier” means thesmallest dimension of a synthetic nanocarrier measured along any axis ofthe synthetic nanocarrier. For example, for a spheroidal syntheticnanocarrier, the maximum and minimum dimension of a syntheticnanocarrier would be substantially identical, and would be the size ofits diameter. Similarly, for a cuboidal synthetic nanocarrier, theminimum dimension of a synthetic nanocarrier would be the smallest ofits height, width or length, while the maximum dimension of a syntheticnanocarrier would be the largest of its height, width or length. In anembodiment, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is equal to or greater than 100 nm. In an embodiment, a maximumdimension of at least 75%, preferably at least 80%, more preferably atleast 90%, of the synthetic nanocarriers in a sample, based on the totalnumber of synthetic nanocarriers in the sample, is equal to or less than5 μm. Preferably, a minimum dimension of at least 75%, preferably atleast 80%, more preferably at least 90%, of the synthetic nanocarriersin a sample, based on the total number of synthetic nanocarriers in thesample, is greater than 110 nm, more preferably greater than 120 nm,more preferably greater than 130 nm, and more preferably still greaterthan 150 nm. Aspects ratios of the maximum and minimum dimensions ofinventive synthetic nanocarriers may vary depending on the embodiment.For instance, aspect ratios of the maximum to minimum dimensions of thesynthetic nanocarriers may vary from 1:1 to 1,000,000:1, preferably from1:1 to 100,000:1, more preferably from 1:1 to 10,000:1, more preferablyfrom 1:1 to 1000:1, still more preferably from 1:1 to 100:1, and yetmore preferably from 1:1 to 10:1. Preferably, a maximum dimension of atleast 75%, preferably at least 80%, more preferably at least 90%, of thesynthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample is equal to or less than 3 μm, morepreferably equal to or less than 2 μm, more preferably equal to or lessthan 1 μm, more preferably equal to or less than 800 nm, more preferablyequal to or less than 600 nm, and more preferably still equal to or lessthan 500 nm. In preferred embodiments, a minimum dimension of at least75%, preferably at least 80%, more preferably at least 90%, of thesynthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample, is equal to or greater than 100nm, more preferably equal to or greater than 120 nm, more preferablyequal to or greater than 130 nm, more preferably equal to or greaterthan 140 nm, and more preferably still equal to or greater than 150 nm.Measurement of synthetic nanocarrier dimensions (e.g., diameter) isobtained by suspending the synthetic nanocarriers in a liquid (usuallyaqueous) media and using dynamic light scattering (DLS) (e.g. using aBrookhaven ZetaPALS instrument). For example, a suspension of syntheticnanocarriers can be diluted from an aqueous buffer into purified waterto achieve a final synthetic nanocarrier suspension concentration ofapproximately 0.01 to 0.1 mg/mL. The diluted suspension may be prepareddirectly inside, or transferred to, a suitable cuvette for DLS analysis.The cuvette may then be placed in the DLS, allowed to equilibrate to thecontrolled temperature, and then scanned for sufficient time to acquirea stable and reproducible distribution based on appropriate inputs forviscosity of the medium and refractive indicies of the sample. Theeffective diameter, or mean of the distribution, is then reported.“Dimension” or “size” or “diameter” of synthetic nanocarriers means themean of a particle size distribution obtained using dynamic lightscattering.

“MHC” refers to major histocompatibility complex, a large genomic regionor gene family found in most vertebrates that encodes MHC molecules thatdisplay fragments or epitopes of processed proteins on the cell surface.The presentation of MHC:peptide on cell surfaces allows for surveillanceby immune cells, usually a T cell. There are two general classes of MHCmolecules: Class I and Class II. Generally, Class I MHC molecules arefound on nucleated cells and present peptides to cytotoxic T cells.Class II MHC molecules are found on certain immune cells, chieflymacrophages, B cells and dendritic cells, collectively known asprofessional APCs. The best-known genes in the MHC region are the subsetthat encodes antigen-presenting proteins on the cell surface. In humans,these genes are referred to as human leukocyte antigen (HLA) genes.

“Non-methoxy-terminated polymer” means a polymer that has at least oneterminus that ends with a moiety other than methoxy. In someembodiments, the polymer has at least two termini that ends with amoiety other than methoxy. In other embodiments, the polymer has notermini that ends with methoxy. “Non-methoxy-terminated, pluronicpolymer” means a polymer other than a linear pluronic polymer withmethoxy at both termini. Polymeric nanoparticles as provided herein cancomprise non-methoxy-terminated polymers or non-methoxy-terminated,pluronic polymers.

“Obtained” means taken directly from a material and used withsubstantially no modification and/or processing.

“Pharmaceutically acceptable excipient” means a pharmacologicallyinactive material used together with the recited synthetic nanocarriersto formulate the inventive compositions. Pharmaceutically acceptableexcipients comprise a variety of materials known in the art, includingbut not limited to saccharides (such as glucose, lactose, and the like),preservatives such as antimicrobial agents, reconstitution aids,colorants, saline (such as phosphate buffered saline), and buffers.

“Protocol” refers to any dosing regimen of one or more substances to asubject. A dosing regimen may include the amount, frequency and/or modeof administration. In some embodiments, such a protocol may be used toadminister one or more compositions of the invention to one or more testsubjects. Immune responses in these test subject can then be assessed todetermine whether or not the protocol was effective in reducing anundesired immune response or generating a desired immune response (e.g.,the promotion of a tolerogenic effect). Any other therapeutic and/orprophylactic effect may also be assessed instead of or in addition tothe aforementioned immune responses. Whether or not a protocol had adesired effect can be determined using any of the methods providedherein or otherwise known in the art. For example, a population of cellsmay be obtained from a subject to which a composition provided hereinhas been administered according to a specific protocol in order todetermine whether or not specific immune cells, cytokines, antibodies,etc. were reduced, generated, activated, etc. Useful methods fordetecting the presence and/or number of immune cells include, but arenot limited to, flow cytometric methods (e.g., FACS) andimmunohistochemistry methods. Antibodies and other binding agents forspecific staining of immune cell markers, are commercially available.Such kits typically include staining reagents for multiple antigens thatallow for FACS-based detection, separation and/or quantitation of adesired cell population from a heterogeneous population of cells.

“Providing a subject” is any action or set of actions that causes aclinician to come in contact with a subject and administer a compositionprovided herein thereto or to perform a method provided hereinthereupon. Preferably, the subject is one who is in need of atolerogenic immune response as provided herein. The action or set ofactions may be either directly oneself or indirectly, such as, but notlimited to, an unrelated third party that takes an action throughreliance on one's words or deeds.

“Subject” means animals, including warm blooded mammals such as humansand primates; avians; domestic household or farm animals such as cats,dogs, sheep, goats, cattle, horses and pigs; laboratory animals such asmice, rats and guinea pigs; fish; reptiles; zoo and wild animals; andthe like.

“Substantially no B cell epitopes” refers to the absence of B cellepitopes in an amount (by itself, within the context of the antigen, inconjunction with a carrier or in conjunction with an inventivecomposition) that stimulates substantial activation of a B cellresponse. In embodiments, a composition with substantially no B cellepitopes does not contain a measurable amount of B cell epitopes of anantigen. In other embodiments, such a composition may comprise ameasurable amount of B cell epitopes of an antigen but said amount isnot effective to generate a measurable B cell immune response (byitself, within the context of the antigen, in conjunction with a carrieror in conjunction with an inventive composition), such asantigen-specific antibody production or antigen-specific B cellproliferation and/or activity, or is not effective to generate asignificant measurable B cell immune response (by itself, within thecontext of the antigen, in conjunction with a carrier or in conjunctionwith an inventive composition). In some embodiments, a significantmeasurable B cell immune response is one that produces or would beexpected to produce an adverse clinical result in a subject. In otherembodiments, a significant measurable B cell immune response is one thatis greater than the level of the same type of immune response (e.g.,antigen-specific antibody production or antigen-specific B cellproliferation and/or activity) produced by a control antigen (e.g., oneknown not to comprise B cell epitopes of the antigen or to stimulate Bcell immune responses). In some embodiments, a significant measurable Bcell immune response, such as a measurement of antibody titers (e.g., byELISA) is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 15-fold, 20-fold or more greater than the same type ofresponse produced by a control (e.g., control antigen). In otherembodiments, a composition with substantially no B cell epitopes is onethat produces little to no antigen-specific antibody titers (by itself,within the context of the antigen, in conjunction with a carrier or inconjunction with an inventive composition). Such compositions includethose that produce an antibody titer (as an EC50 value) of less than500, 400, 300, 200, 100, 50, 40, 30, 20 or 10. In other embodiments, asignificant measurable B cell immune response, is a measurement of thenumber or proliferation of B cells that is 10%, 25%, 50%, 100%, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,15-fold, 20-fold or more greater that the same type of response producedby a control. Other methods for measuring B cell responses are known tothose of ordinary skill in the art.

In embodiments, to ensure that a composition comprises substantially noB cell epitopes, antigens are selected such that they do not comprise Bcell epitopes for coupling to the synthetic nanocarriers as providedherein. In other embodiments, to ensure that a composition comprisessubstantially no B cell epitopes of an antigen, the syntheticnanocarriers coupled to the antigen are produced and tested for B cellimmune responses (e.g., antigen-specific antibody production, B cellproliferation and/or activity). Compositions that exhibit the desiredproperties may then be selected.

“Synthetic nanocarrier(s)” means a discrete object that is not found innature, and that possesses at least one dimension that is less than orequal to 5 microns in size. Albumin nanoparticles are generally includedas synthetic nanocarriers, however in certain embodiments the syntheticnanocarriers do not comprise albumin nanoparticles. In embodiments,inventive synthetic nanocarriers do not comprise chitosan. In otherembodiments, inventive synthetic nanocarriers are not lipid-basednanoparticles. In further embodiments, inventive synthetic nanocarriersdo not comprise a phospholipid.

A synthetic nanocarrier can be, but is not limited to, one or aplurality of lipid-based nanoparticles (also referred to herein as lipidnanoparticles, i.e., nanoparticles where the majority of the materialthat makes up their structure are lipids), polymeric nanoparticles,metallic nanoparticles, surfactant-based emulsions, dendrimers,buckyballs, nanowires, virus-like particles (i.e., particles that areprimarily made up of viral structural proteins but that are notinfectious or have low infectivity), peptide or protein-based particles(also referred to herein as protein particles, i.e., particles where themajority of the material that makes up their structure are peptides orproteins) (such as albumin nanoparticles) and/or nanoparticles that aredeveloped using a combination of nanomaterials such as lipid-polymernanoparticles. Synthetic nanocarriers may be a variety of differentshapes, including but not limited to spheroidal, cuboidal, pyramidal,oblong, cylindrical, toroidal, and the like. Synthetic nanocarriersaccording to the invention comprise one or more surfaces. Exemplarysynthetic nanocarriers that can be adapted for use in the practice ofthe present invention comprise: (1) the biodegradable nanoparticlesdisclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymericnanoparticles of Published US Patent Application 20060002852 to Saltzmanet al., (3) the lithographically constructed nanoparticles of PublishedUS Patent Application 20090028910 to DeSimone et al., (4) the disclosureof WO 2009/051837 to von Andrian et al., (5) the nanoparticles disclosedin Published US Patent Application 2008/0145441 to Penades et al., (6)the protein nanoparticles disclosed in Published US Patent Application20090226525 to de los Rios et al., (7) the virus-like particlesdisclosed in published US Patent Application 20060222652 to Sebbel etal., (8) the nucleic acid coupled virus-like particles disclosed inpublished US Patent Application 20060251677 to Bachmann et al., (9) thevirus-like particles disclosed in WO2010047839A1 or WO2009106999A2, (10)the nanoprecipitated nanoparticles disclosed in P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010), or (11) apoptotic cells, apoptotic bodies or the synthetic orsemisynthetic mimics disclosed in U.S. Publication 2002/0086049. Inembodiments, synthetic nanocarriers may possess an aspect ratio greaterthan 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.

Synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface with hydroxyl groups thatactivate complement or alternatively comprise a surface that consistsessentially of moieties that are not hydroxyl groups that activatecomplement. In a preferred embodiment, synthetic nanocarriers accordingto the invention that have a minimum dimension of equal to or less thanabout 100 nm, preferably equal to or less than 100 nm, do not comprise asurface that substantially activates complement or alternativelycomprise a surface that consists essentially of moieties that do notsubstantially activate complement. In a more preferred embodiment,synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface that activates complement oralternatively comprise a surface that consists essentially of moietiesthat do not activate complement. In embodiments, synthetic nanocarriersexclude virus-like particles. In embodiments, synthetic nanocarriers maypossess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5,1:7, or greater than 1:10.

“T cell antigen” means a CD4+ T-cell antigen or CD8+ cell antigen. “CD4+T-cell antigen” means any antigen that is recognized by and triggers animmune response in a CD4+ T-cell e.g., an antigen that is specificallyrecognized by a T-cell receptor on a CD4+ T cell via presentation of theantigen or portion thereof bound to a Class II major histocompatabilitycomplex molecule (MHC). “CD8+ T cell antigen” means any antigen that isrecognized by and triggers an immune response in a CD8+ T-cell e.g., anantigen that is specifically recognized by a T-cell receptor on a CD8+ Tcell via presentation of the antigen or portion thereof bound to a ClassI major histocompatability complex molecule (MHC). In some embodiments,an antigen that is a T cell antigen is also a B cell antigen. In otherembodiments, the T cell antigen is not also a B cell antigen. T cellantigens generally are proteins or peptides.

A “therapeutic protein” refers to any protein or protein-based therapythat may be administered to a subject and have a therapeutic effect.Such therapies include protein replacement and protein supplementationtherapies. Such therapies also include the administration of exogenousor foreign protein, antibody therapies, and cell or cell-basedtherapies. Therapeutic proteins include enzymes, enzyme cofactors,hormones, blood clotting factors, cytokines, growth factors, monoclonalantibodies and polyclonal antibodies. Examples of other therapeuticproteins are provided elsewhere herein. Therapeutic proteins may beproduced in, on or by cells and may be obtained from such cells oradministered in the form of such cells. In embodiments, the therapeuticprotein is produced in, on or by mammalian cells, insect cells, yeastcells, bacteria cells, plant cells, transgenic animal cells, transgenicplant cells, etc. The therapeutic protein may be recombinantly producedin such cells. The therapeutic protein may be produced in, on or by avirally transformed cell. The therapeutic protein may also be producedin, on or by autologous cells that have been transfected, transduced orotherwise manipulated to express it. Alternatively, the therapeuticprotein may be administered as a nucleic acid or by introducing anucleic acid into a virus, VLP, liposome, etc. Alternatively, thetherapeutic protein may be obtained from such forms and administered asthe therapeutic protein itself. Subjects, therefore, include any subjectthat has received, is receiving or will receive any of the foregoing.Such subject includes subjects that have received, is receiving or willreceive gene therapy, autologous cells that have been transfected,transduced or otherwise manipulated to express a therapeutic protein,polypeptide or peptide; or cells that express a therapeutic protein,polypeptide or peptide.

“Therapeutic protein APC presentable antigen” means an antigen that isassociated with a therapeutic protein (i.e., the therapeutic protein ora fragment thereof can generate an immune response against thetherapeutic protein (e.g., the production of therapeuticprotein-specific antibodies)). Generally, therapeutic protein APCpresentable antigens can be presented for recognition by the immunesystem (e.g., cells of the immune system, such as presented by antigenpresenting cells, including but not limited to dendritic cells, B cellsor macrophages). The therapeutic protein APC presentable antigen can bepresented for recognition by, for example, T cells. Such antigens may berecognized by and trigger an immune response in a T cell viapresentation of an epitope of the antigen bound to a Class I or Class IImajor histocompatability complex molecule (MHC). Therapeutic protein APCpresentable antigens generally include proteins, polypeptides, peptides,lipoproteins, or are contained or expressed in, on or by cells. Thetherapeutic protein antigens, in some embodiments, are coupled tosynthetic nanocarriers and comprise MHC Class I-restricted epitopesand/or MHC Class II-restricted epitopes and/or B cell epitopes. In otherembodiments, the antigens do not comprise B cell epitopes, such as whenthe inclusion of the B cell epitopes would exacerbate an undesiredimmune response. In other embodiments, the antigens comprise MHC ClassII-restricted epitopes and substantially no B cell epitopes. Preferably,one or more tolerogenic immune responses specific to the therapeuticprotein result with the compositions provided herein.

“Tolerogenic immune response” means any immune response that can lead toimmune suppression specific to an antigen or a cell, tissue, organ, etc.that expresses such an antigen. Such immune responses include anyreduction, delay or inhibition in an undesired immune response specificto the antigen or cell, tissue, organ, etc. that expresses such antigen.Such immune responses also include any stimulation, production,induction, promotion or recruitment in a desired immune responsespecific to the antigen or cell, tissue, organ, etc. that expresses suchantigen. Tolerogenic immune responses, therefore, include the absence ofor reduction in an undesired immune response to an antigen that can bemediated by antigen reactive cells as well as the presence or promotionof suppressive cells. Tolerogenic immune responses as provided hereininclude immunological tolerance. To “generate a tolerogenic immuneresponse” refers to the generation of any of the foregoing immuneresponses specific to an antigen or cell, tissue, organ, etc. thatexpresses such antigen. The tolerogenic immune response can be theresult of MHC Class I-restricted presentation and/or MHC ClassII-restricted presentation and/or B cell presentation and/orpresentation by CD1d, etc.

Tolerogenic immune responses include any reduction, delay or inhibitionin CD4+ T cell, CD8+ T cell or B cell proliferation and/or activity.Tolerogenic immune responses also include a reduction inantigen-specific antibody production. Tolerogenic immune responses canalso include any response that leads to the stimulation, induction,production or recruitment of regulatory cells, such as CD4+ Treg cells,CD8+ Treg cells, Breg cells, etc. In some embodiments, the tolerogenicimmune response, is one that results in the conversion to a regulatoryphenotype characterized by the production, induction, stimulation orrecruitment of regulatory cells.

Tolerogenic immune responses also include any response that leads to thestimulation, production or recruitment of CD4+ Treg cells and/or CD8+Treg cells. CD4+ Treg cells can express the transcription factor FoxP3and inhibit inflammatory responses and auto-immune inflammatory diseases(Human regulatory T cells in autoimmune diseases. Cvetanovich G L,Hafler D A. Curr Opin Immunol. 2010 December; 22(6):753-60. Regulatory Tcells and autoimmunity. Vila J, Isaacs J D, Anderson A E. Curr OpinHematol. 2009 July; 16(4):274-9). Such cells also suppress T-cell helpto B-cells and induce tolerance to both self and foreign antigens(Therapeutic approaches to allergy and autoimmunity based on FoxP3+regulatory T-cell activation and expansion. Miyara M, Wing K, SakaguchiS. J Allergy Clin Immunol. 2009 April; 123(4):749-55). CD4+ Treg cellsrecognize antigen when presented by Class II proteins on APCs. CD8+ Tregcells, which recognize antigen presented by Class I (and Qa-1), can alsosuppress T-cell help to B-cells and result in activation ofantigen-specific suppression inducing tolerance to both self and foreignantigens. Disruption of the interaction of Qa-1 with CD8+ Treg cells hasbeen shown to dysregulate immune responses and results in thedevelopment of auto-antibody formation and an auto-immune lethalsystemic-lupus-erythematosus (Kim et al., Nature. 2010 Sep. 16, 467(7313): 328-32). CD8+ Treg cells have also been shown to inhibit modelsof autoimmune inflammatory diseases including rheumatoid arthritis andcolitis (CD4+CD25+ regulatory T cells in autoimmune arthritis. Oh S,Rankin A L, Caton A J. Immunol Rev. 2010 January; 233(1):97-111.Regulatory T cells in inflammatory bowel disease. Boden E K, Snapper SB. Curr Opin Gastroenterol. 2008 November; 24(6):733-41). In someembodiments, the compositions provided can effectively result in bothtypes of responses (CD4+ Treg and CD8+ Treg). In other embodiments,FoxP3 can be induced in other immune cells, such as macrophages, iNKTcells, etc., and the compositions provided herein can result in one ormore of these responses as well.

Tolerogenic immune responses also include, but are not limited to, theinduction of regulatory cytokines, such as Treg cytokines; induction ofinhibitory cytokines; the inhibition of inflammatory cytokines (e.g.,IL-4, IL-1b, IL-5, TNF-α, IL-6, GM-CSF, IFN-γ, IL-2, IL-9, IL-12, IL-17,IL-18, IL-21, IL-22, IL-23, M-CSF, C reactive protein, acute phaseprotein, chemokines (e.g., MCP-1, RANTES, MIP-1α, MIP-1β, MIG, ITAC orIP-10), the production of anti-inflammatory cytokines (e.g., IL-4,IL-13, IL-10, etc.), chemokines (e.g., CCL-2, CXCL8), proteases (e.g.,MMP-3, MMP-9), leukotrienes (e.g., CysLT-1, CysLT-2), prostaglandins(e.g., PGE2) or histamines; the inhibition of polarization to a Th17,Th1 or Th2 immune response; the inhibition of effector cell-specificcytokines: Th17 (e.g., IL-17, IL-25), Th1 (IFN-γ), Th2 (e.g., IL-4,IL-13); the inhibition of Th1-, Th2- or TH17-specific transcriptionfactors; the inhibition of proliferation of effector T cells; theinduction of apoptosis of effector T cells; the induction of tolerogenicdendritic cell-specific genes, the induction of FoxP3 expression, theinhibition of IgE induction or IgE-mediated immune responses; theinhibition of antibody responses (e.g., antigen-specific antibodyproduction); the inhibition of T helper cell response; the production ofTGF-β and/or IL-10; the inhibition of effector function ofautoantibodies (e.g., inhibition in the depletion of cells, cell ortissue damage or complement activation); etc.

Any of the foregoing may be measured in vivo in one or more animalmodels or may be measured in vitro. One of ordinary skill in the art isfamiliar with such in vivo or in vitro measurements. Undesired immuneresponses or tolerogenic immune responses can be monitored using, forexample, methods of assessing immune cell number and/or function,tetramer analysis, ELISPOT, flow cytometry-based analysis of cytokineexpression, cytokine secretion, cytokine expression profiling, geneexpression profiling, protein expression profiling, analysis of cellsurface markers, PCR-based detection of immune cell receptor gene usage(see T. Clay et al., “Assays for Monitoring Cellular Immune Response toActive Immunotherapy of Cancer” Clinical Cancer Research 7:1127-1135(2001)), etc. Undesired immune responses or tolerogenic immune responsesmay also be monitored using, for example, methods of assessing proteinlevels in plasma or serum, immune cell proliferation and/or functionalassays, etc. In some embodiments, tolerogenic immune responses can bemonitored by assessing the induction of FoxP3. In addition, specificmethods are described in more detail in the Examples.

Preferably, tolerogenic immune responses lead to the inhibition of thedevelopment, progression or pathology of the diseases, disorders orconditions described herein. Whether or not the inventive compositionscan lead to the inhibition of the development, progression or pathologyof the diseases, disorders or conditions described herein can bemeasured with animal models of such diseases, disorders or conditions.In some embodiments, the reduction of an undesired immune response orgeneration of a tolerogenic immune response may be assessed bydetermining clinical endpoints, clinical efficacy, clinical symptoms,disease biomarkers and/or clinical scores. Undesired immune responses ortolerogenic immune responses can also be assessed with diagnostic teststo assess the presence or absence of a disease, disorder or condition asprovided herein. Undesired immune responses can further be assessed bymethods of measuring therapeutic proteins levels and/or function in asubject.

In some embodiments, monitoring or assessing the generation of anundesired immune response or a tolerogenic immune response in a subjectcan be prior to the administration of a composition of syntheticnanocarriers provided herein and/or prior to administration of atherapeutic protein. In other embodiments, assessing the generation ofan undesired immune response or tolerogenic immune response can be afteradministration of a composition of synthetic nanocarriers providedherein and/or and after administration of a therapeutic protein. In someembodiments, the assessment is done after administration of thecomposition of synthetic nanocarriers, but prior to administration ofthe therapeutic protein. In other embodiments, the assessment is doneafter administration of the therapeutic protein, but prior toadministration of the composition. In still other embodiments, theassessment is performed prior to both the administration of thesynthetic nanocarriers and the therapeutic protein, while in yet otherembodiments the assessment is performed after administration of both thesynthetic nanocarriers and the therapeutic protein. In furtherembodiments, the assessment is performed both prior to and after theadministration of the synthetic nanocarriers and/or the therapeuticprotein. In still other embodiments, the assessment is performed morethan once on the subject to determine that a desirable immune state ismaintained in the subject, such as a subject that has been, is being orwill be administered a therapeutic protein.

An antibody response can be assessed by determining one or more antibodytiters. “Antibody titer” means a measurable level of antibodyproduction. Methods for measuring antibody titers are known in the artand include Enzyme-linked Immunosorbent Assay (ELISA). In embodiments,the antibody response can be quantitated, for example, as the number ofantibodies, concentration of antibodies or titer. The values can beabsolute or they can be relative. Assays for quantifying an antibodyresponse include antibody capture assays, enzyme-linked immunosorbentassays (ELISAs), inhibition liquid phase absorption assays (ILPAAs),rocket immunoelectrophoresis (RIE) assays and line immunoelectrophoresis(LIE) assays. When an antibody response is compared to another antibodyresponse the same type of quantitative value (e.g., titer) and method ofmeasurement (e.g., ELISA) is preferably used to make the comparison.

An ELISA method for measuring an antibody titer, for example, a typicalsandwich ELISA, may consist of the following steps (i) preparing anELISA-plate coating material such that the antibody target of interestis coupled to a substrate polymer or other suitable material (ii)preparing the coating material in an aqueous solution (such as PBS) anddelivering the coating material solution to the wells of a multiwellplate for overnight deposition of the coating onto the multiwell plate(iii) thoroughly washing the multiwell plate with wash buffer (such as0.05% Tween-20 in PBS) to remove excess coating material (iv) blockingthe plate for nonspecific binding by applying a diluent solution (suchas 10% fetal bovine serum in PBS), (v) washing the blocking/diluentsolution from the plate with wash buffer (vi) diluting the serumsample(s) containing antibodies and appropriate standards (positivecontrols) with diluent as required to obtain a concentration thatsuitably saturates the ELISA response (vii) serially diluting the plasmasamples on the multiwell plate such to cover a range of concentrationssuitable for generating an ELISA response curve (viii) incubating theplate to provide for antibody-target binding (ix) washing the plate withwash buffer to remove antibodies not bound to antigen (x) adding anappropriate concentration of a secondary detection antibody in samediluent such as a biotin-coupled detection antibody capable of bindingthe primary antibody (xi) incubating the plate with the applieddetection antibody, followed by washing with wash buffer (xii) adding anenzyme such as streptavidin-HRP (horse radish peroxidase) that will bindto biotin found on biotinylated antibodies and incubating (xiii) washingthe multiwell plate (xiv) adding substrate(s) (such as TMB solution) tothe plate (xv) applying a stop solution (such as 2N sulfuric acid) whencolor development is complete (xvi) reading optical density of the platewells at a specific wavelength for the substrate (450 nm withsubtraction of readings at 570 nm) (xvi) applying a suitablemultiparameter curve fit to the data and defining half-maximal effectiveconcentration (EC50) as the concentration on the curve at which half themaximum OD value for the plate standards is achieved.

“Undesired immune response” refers to any undesired immune response thatresults from exposure to an antigen, promotes or exacerbates a disease,disorder or condition provided herein (or a symptom thereof), or issymptomatic of a disease, disorder or condition provided herein. Suchimmune responses generally have a negative impact on a subject's healthor is symptomatic of a negative impact on a subject's health. Undesiredimmune responses include antigen-specific antibody production,antigen-specific B cell proliferation and/or activity orantigen-specific CD4+ T cell proliferation and/or activity.

C. INVENTIVE COMPOSITIONS

Provided herein are tolerogenic synthetic nanocarrier compositionscomprising immunosuppressants and therapeutic protein APC presentableantigens and related methods. Such compositions and methods are usefulfor reducing the generation of undesired immune responses and promotingthe generation of tolerogenic immune responses that are specific totherapeutic proteins. The compositions can be administered to subjectsin which a tolerogenic immune response to therapeutic proteins isdesired. Such subjects include those that have been, are being or willbe administered a therapeutic protein. In embodiments, a subject isadministered the synthetic nanocarriers provided herein and is thenadministered one or more therapeutic proteins. In other embodiments, asubject is administered a therapeutic protein and then the syntheticnanocarriers provided. In still other embodiments, the syntheticnanocarriers and the one or more therapeutic proteins are administeredconcomitantly.

As mentioned above, the synthetic nanocarriers are designed to compriseimmunosuppressants and, in some embodiments, antigen against which atolerogenic effect is desired. In embodiments, the antigens comprise MHCClass II-restricted epitopes that when presented in conjunction withimmunosuppressant can lead to tolerogenic effects, such as the reductionin antigen-specific CD4+ T cell proliferation and/or activity. Theresulting tolerogenic effects also include a reduction inantigen-specific B cell proliferation and/or activity and/or a reductionin antigen-specific antibody production. A wide variety of syntheticnanocarriers can be used according to the invention. In someembodiments, synthetic nanocarriers are spheres or spheroids. In someembodiments, synthetic nanocarriers are flat or plate-shaped. In someembodiments, synthetic nanocarriers are cubes or cubic. In someembodiments, synthetic nanocarriers are ovals or ellipses. In someembodiments, synthetic nanocarriers are cylinders, cones, or pyramids.

In some embodiments, it is desirable to use a population of syntheticnanocarriers that is relatively uniform in terms of size, shape, and/orcomposition so that each synthetic nanocarrier has similar properties.For example, at least 80%, at least 90%, or at least 95% of thesynthetic nanocarriers, based on the total number of syntheticnanocarriers, may have a minimum dimension or maximum dimension thatfalls within 5%, 10%, or 20% of the average diameter or averagedimension of the synthetic nanocarriers. In some embodiments, apopulation of synthetic nanocarriers may be heterogeneous with respectto size, shape, and/or composition.

Synthetic nanocarriers can be solid or hollow and can comprise one ormore layers. In some embodiments, each layer has a unique compositionand unique properties relative to the other layer(s). To give but oneexample, synthetic nanocarriers may have a core/shell structure, whereinthe core is one layer (e.g. a polymeric core) and the shell is a secondlayer (e.g. a lipid bilayer or monolayer). Synthetic nanocarriers maycomprise a plurality of different layers.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more lipids. In some embodiments, a synthetic nanocarrier maycomprise a liposome. In some embodiments, a synthetic nanocarrier maycomprise a lipid bilayer. In some embodiments, a synthetic nanocarriermay comprise a lipid monolayer. In some embodiments, a syntheticnanocarrier may comprise a micelle. In some embodiments, a syntheticnanocarrier may comprise a core comprising a polymeric matrix surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In someembodiments, a synthetic nanocarrier may comprise a non-polymeric core(e.g., metal particle, quantum dot, ceramic particle, bone particle,viral particle, proteins, nucleic acids, carbohydrates, etc.) surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).

In other embodiments, synthetic nanocarriers may comprise metalparticles, quantum dots, ceramic particles, etc. In some embodiments, anon-polymeric synthetic nanocarrier is an aggregate of non-polymericcomponents, such as an aggregate of metal atoms (e.g., gold atoms).

In some embodiments, synthetic nanocarriers may optionally comprise oneor more amphiphilic entities. In some embodiments, an amphiphilic entitycan promote the production of synthetic nanocarriers with increasedstability, improved uniformity, or increased viscosity. In someembodiments, amphiphilic entities can be associated with the interiorsurface of a lipid membrane (e.g., lipid bilayer, lipid monolayer,etc.). Many amphiphilic entities known in the art are suitable for usein making synthetic nanocarriers in accordance with the presentinvention. Such amphiphilic entities include, but are not limited to,phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine(DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides;sorbitan trioleate (Span®85) glycocholate; sorbitan monolaurate(Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60);polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85(Tween®85); polyoxyethylene monostearate; surfactin; a poloxomer; asorbitan fatty acid ester such as sorbitan trioleate; lecithin;lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)400-monostearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; ion pairing agents; and combinationsthereof. An amphiphilic entity component may be a mixture of differentamphiphilic entities. Those skilled in the art will recognize that thisis an exemplary, not comprehensive, list of substances with surfactantactivity. Any amphiphilic entity may be used in the production ofsynthetic nanocarriers to be used in accordance with the presentinvention.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more carbohydrates. Carbohydrates may be natural or synthetic. Acarbohydrate may be a derivatized natural carbohydrate. In certainembodiments, a carbohydrate comprises monosaccharide or disaccharide,including but not limited to glucose, fructose, galactose, ribose,lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose,arabinose, glucoronic acid, galactoronic acid, mannuronic acid,glucosamine, galatosamine, and neuramic acid. In certain embodiments, acarbohydrate is a polysaccharide, including but not limited to pullulan,cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose(HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran,cyclodextran, glycogen, hydroxyethylstarch, carageenan, glycon, amylose,chitosan, N,O-carboxylmethylchitosan, algin and alginic acid, starch,chitin, inulin, konjac, glucommannan, pustulan, heparin, hyaluronicacid, curdlan, and xanthan. In embodiments, the inventive syntheticnanocarriers do not comprise (or specifically exclude) carbohydrates,such as a polysaccharide. In certain embodiments, the carbohydrate maycomprise a carbohydrate derivative such as a sugar alcohol, includingbut not limited to mannitol, sorbitol, xylitol, erythritol, maltitol,and lactitol.

In some embodiments, synthetic nanocarriers can comprise one or morepolymers. In some embodiments, the synthetic nanocarriers comprise oneor more polymers that is a non-methoxy-terminated, pluronic polymer. Insome embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or99% (weight/weight) of the polymers that make up the syntheticnanocarriers are non-methoxy-terminated, pluronic polymers. In someembodiments, all of the polymers that make up the synthetic nanocarriersare non-methoxy-terminated, pluronic polymers. In some embodiments, thesynthetic nanocarriers comprise one or more polymers that is anon-methoxy-terminated polymer. In some embodiments, at least 1%, 2%,3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of thepolymers that make up the synthetic nanocarriers arenon-methoxy-terminated polymers. In some embodiments, all of thepolymers that make up the synthetic nanocarriers arenon-methoxy-terminated polymers. In some embodiments, the syntheticnanocarriers comprise one or more polymers that do not comprise pluronicpolymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, or 99% (weight/weight) of the polymers that make up thesynthetic nanocarriers do not comprise pluronic polymer. In someembodiments, all of the polymers that make up the synthetic nanocarriersdo not comprise pluronic polymer. In some embodiments, such a polymercan be surrounded by a coating layer (e.g., liposome, lipid monolayer,micelle, etc.). In some embodiments, various elements of the syntheticnanocarriers can be coupled with the polymer.

The immunosuppressants and/or antigens can be coupled to the syntheticnanocarriers by any of a number of methods. Generally, the coupling canbe a result of bonding between the immunosuppressants and/or antigensand the synthetic nanocarriers. This bonding can result in theimmunosuppressants and/or antigens being attached to the surface of thesynthetic nanocarriers and/or contained within (encapsulated) thesynthetic nanocarriers. In some embodiments, however, theimmunosuppressants and/or antigens are encapsulated by the syntheticnanocarriers as a result of the structure of the synthetic nanocarriersrather than bonding to the synthetic nanocarriers. In preferableembodiments, the synthetic nanocarrier comprises a polymer as providedherein, and the immunosuppressants and/or antigens are coupled to thepolymer.

When coupling occurs as a result of bonding between theimmunosuppressants and/or antigens and synthetic nanocarriers, thecoupling may occur via a coupling moiety. A coupling moiety can be anymoiety through which an immunosuppressant and/or antigen is bonded to asynthetic nanocarrier. Such moieties include covalent bonds, such as anamide bond or ester bond, as well as separate molecules that bond(covalently or non-covalently) the immunosuppressant and/or antigen tothe synthetic nanocarrier. Such molecules include linkers or polymers ora unit thereof. For example, the coupling moiety can comprise a chargedpolymer to which an immunosuppressant and/or antigen electrostaticallybinds. As another example, the coupling moiety can comprise a polymer orunit thereof to which it is covalently bonded.

In preferred embodiments, the synthetic nanocarriers comprise a polymeras provided herein. These synthetic nanocarriers can be completelypolymeric or they can be a mix of polymers and other materials.

In some embodiments, the polymers of a synthetic nanocarrier associateto form a polymeric matrix. In some of these embodiments, a component,such as an immunosuppressant or antigen, can be covalently associatedwith one or more polymers of the polymeric matrix. In some embodiments,covalent association is mediated by a linker. In some embodiments, acomponent can be noncovalently associated with one or more polymers ofthe polymeric matrix. For example, in some embodiments, a component canbe encapsulated within, surrounded by, and/or dispersed throughout apolymeric matrix. Alternatively or additionally, a component can beassociated with one or more polymers of a polymeric matrix byhydrophobic interactions, charge interactions, van der Waals forces,etc. A wide variety of polymers and methods for forming polymericmatrices therefrom are known conventionally.

Polymers may be natural or unnatural (synthetic) polymers. Polymers maybe homopolymers or copolymers comprising two or more monomers. In termsof sequence, copolymers may be random, block, or comprise a combinationof random and block sequences. Typically, polymers in accordance withthe present invention are organic polymers.

In some embodiments, the polymer comprises a polyester, polycarbonate,polyamide, or polyether, or unit thereof. In other embodiments, thepolymer comprises poly(ethylene glycol) (PEG), polypropylene glycol,poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid),or a polycaprolactone, or unit thereof. In some embodiments, it ispreferred that the polymer is biodegradable. Therefore, in theseembodiments, it is preferred that if the polymer comprises a polyether,such as poly(ethylene glycol) or polypropylene glycol or unit thereof,the polymer comprises a block-co-polymer of a polyether and abiodegradable polymer such that the polymer is biodegradable. In otherembodiments, the polymer does not solely comprise a polyether or unitthereof, such as poly(ethylene glycol) or polypropylene glycol or unitthereof.

Other examples of polymers suitable for use in the present inventioninclude, but are not limited to polyethylenes, polycarbonates (e.g.poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic anhydride)),polypropylfumerates, polyamides (e.g. polycaprolactam), polyacetals,polyethers, polyesters (e.g., polylactide, polyglycolide,polylactide-co-glycolide, polycaprolactone, polyhydroxy acid (e.g.poly(β-hydroxyalkanoate))), poly(orthoesters), polycyanoacrylates,polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine,polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

In some embodiments, polymers in accordance with the present inventioninclude polymers which have been approved for use in humans by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. §177.2600, includingbut not limited to polyesters (e.g., polylactic acid,poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymersmay comprise anionic groups (e.g., phosphate group, sulphate group,carboxylate group); cationic groups (e.g., quaternary amine group); orpolar groups (e.g., hydroxyl group, thiol group, amine group). In someembodiments, a synthetic nanocarrier comprising a hydrophilic polymericmatrix generates a hydrophilic environment within the syntheticnanocarrier. In some embodiments, polymers can be hydrophobic. In someembodiments, a synthetic nanocarrier comprising a hydrophobic polymericmatrix generates a hydrophobic environment within the syntheticnanocarrier. Selection of the hydrophilicity or hydrophobicity of thepolymer may have an impact on the nature of materials that areincorporated (e.g. coupled) within the synthetic nanocarrier.

In some embodiments, polymers may be modified with one or more moietiesand/or functional groups. A variety of moieties or functional groups canbe used in accordance with the present invention. In some embodiments,polymers may be modified with polyethylene glycol (PEG), with acarbohydrate, and/or with acyclic polyacetals derived frompolysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Certainembodiments may be made using the general teachings of U.S. Pat. No.5,543,158 to Gref et al., or WO publication WO2009/051837 by Von Andrianet al.

In some embodiments, polymers may be modified with a lipid or fatty acidgroup. In some embodiments, a fatty acid group may be one or more ofbutyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers,PLGA-PEG copolymers, and derivatives thereof. In some embodiments,polyesters include, for example, poly(caprolactone),poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA are characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid:glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention is characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers,polycyanoacrylates, and combinations comprising one or more of theforegoing polymers. The acrylic polymer may comprise fully-polymerizedcopolymers of acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g. DNA, or derivatives thereof).Amine-containing polymers such as poly(lysine) (Zauner et al., 1998,Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, BioconjugateChem., 6:7), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc.Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers(Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897;Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,Bioconjugate Chem., 4:372) are positively-charged at physiological pH,form ion pairs with nucleic acids, and mediate transfection in a varietyof cell lines. In embodiments, the inventive synthetic nanocarriers maynot comprise (or may exclude) cationic polymers.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989,Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633;and Zhou et al., 1990, Macromolecules, 23:3399). Examples of thesepolyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J.Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam etal., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem.Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al.,1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc.,121:5633).

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al.,2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc.,123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J.Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181).More generally, a variety of methods for synthesizing certain suitablepolymers are described in Concise Encyclopedia of Polymer Science andPolymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press,1980; Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al.,Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S.Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used in accordance with the presentinvention without undergoing a cross-linking step. It is further to beunderstood that inventive synthetic nanocarriers may comprise blockcopolymers, graft copolymers, blends, mixtures, and/or adducts of any ofthe foregoing and other polymers. Those skilled in the art willrecognize that the polymers listed herein represent an exemplary, notcomprehensive, list of polymers that can be of use in accordance withthe present invention.

In some embodiments, synthetic nanocarriers do not comprise a polymericcomponent. In some embodiments, synthetic nanocarriers may comprisemetal particles, quantum dots, ceramic particles, etc. In someembodiments, a non-polymeric synthetic nanocarrier is an aggregate ofnon-polymeric components, such as an aggregate of metal atoms (e.g.,gold atoms).

Compositions according to the invention comprise synthetic nanocarriersin combination with pharmaceutically acceptable excipients, such aspreservatives, buffers, saline, or phosphate buffered saline. Thecompositions may be made using conventional pharmaceutical manufacturingand compounding techniques to arrive at useful dosage forms. In anembodiment, inventive synthetic nanocarriers are suspended in sterilesaline solution for injection together with a preservative.

In embodiments, when preparing synthetic nanocarriers as carriers,methods for coupling components to the synthetic nanocarriers may beuseful. If the component is a small molecule it may be of advantage toattach the component to a polymer prior to the assembly of the syntheticnanocarriers. In embodiments, it may also be an advantage to prepare thesynthetic nanocarriers with surface groups that are used to couple thecomponent to the synthetic nanocarrier through the use of these surfacegroups rather than attaching the component to a polymer and then usingthis polymer conjugate in the construction of synthetic nanocarriers.

In certain embodiments, the coupling can be a covalent linker. Inembodiments, peptides according to the invention can be covalentlycoupled to the external surface via a 1,2,3-triazole linker formed bythe 1,3-dipolar cycloaddition reaction of azido groups on the surface ofthe nanocarrier with antigen or immunosuppressant containing an alkynegroup or by the 1,3-dipolar cycloaddition reaction of alkynes on thesurface of the nanocarrier with antigens or immunosuppressantscontaining an azido group. Such cycloaddition reactions are preferablyperformed in the presence of a Cu(I) catalyst along with a suitableCu(I)-ligand and a reducing agent to reduce Cu(II) compound to catalyticactive Cu(I) compound. This Cu(I)-catalyzed azide-alkyne cycloaddition(CuAAC) can also be referred as the click reaction.

Additionally, the covalent coupling may comprise a covalent linker thatcomprises an amide linker, a disulfide linker, a thioether linker, ahydrazone linker, a hydrazide linker, an imine or oxime linker, an ureaor thiourea linker, an amidine linker, an amine linker, and asulfonamide linker.

An amide linker is formed via an amide bond between an amine on onecomponent such as an antigen or immunosuppressant with the carboxylicacid group of a second component such as the nanocarrier. The amide bondin the linker can be made using any of the conventional amide bondforming reactions with suitably protected amino acids and activatedcarboxylic acid such N-hydroxysuccinimide-activated ester.

A disulfide linker is made via the formation of a disulfide (S—S) bondbetween two sulfur atoms of the form, for instance, of R1-S—S—R2. Adisulfide bond can be formed by thiol exchange of a component containingthiol/mercaptan group (—SH) with another activated thiol group on apolymer or nanocarrier or a nanocarrier containing thiol/mercaptangroups with a component containing activated thiol group.

A triazole linker, specifically a 1,2,3-triazole of the form

wherein R1 and R2 may be any chemical entities, is made by the1,3-dipolar cycloaddition reaction of an azide attached to a firstcomponent such as the nanocarrier with a terminal alkyne attached to asecond component such as the immunosuppressant or antigen. The1,3-dipolar cycloaddition reaction is performed with or without acatalyst, preferably with Cu(I)-catalyst, which links the two componentsthrough a 1,2,3-triazole function. This chemistry is described in detailby Sharpless et al., Angew. Chem. Int. Ed. 41(14), 2596, (2002) andMeldal, et al, Chem. Rev., 2008, 108(8), 2952-3015 and is often referredto as a “click” reaction or CuAAC.

In embodiments, a polymer containing an azide or alkyne group, terminalto the polymer chain is prepared. This polymer is then used to prepare asynthetic nanocarrier in such a manner that a plurality of the alkyne orazide groups are positioned on the surface of that nanocarrier.Alternatively, the synthetic nanocarrier can be prepared by anotherroute, and subsequently functionalized with alkyne or azide groups. Thecomponent is prepared with the presence of either an alkyne (if thepolymer contains an azide) or an azide (if the polymer contains analkyne) group. The component is then allowed to react with thenanocarrier via the 1,3-dipolar cycloaddition reaction with or without acatalyst which covalently couples the component to the particle throughthe 1,4-disubstituted 1,2,3-triazole linker.

A thioether linker is made by the formation of a sulfur-carbon(thioether) bond in the form, for instance, of R1-S—R2. Thioether can bemade by either alkylation of a thiol/mercaptan (—SH) group on onecomponent with an alkylating group such as halide or epoxide on a secondcomponent. Thioether linkers can also be formed by Michael addition of athiol/mercaptan group on one component to an electron-deficient alkenegroup on a second component containing a maleimide group or vinylsulfone group as the Michael acceptor. In another way, thioether linkerscan be prepared by the radical thiol-ene reaction of a thiol/mercaptangroup on one component with an alkene group on a second component.

A hydrazone linker is made by the reaction of a hydrazide group on onecomponent with an aldehyde/ketone group on the second component.

A hydrazide linker is formed by the reaction of a hydrazine group on onecomponent with a carboxylic acid group on the second component. Suchreaction is generally performed using chemistry similar to the formationof amide bond where the carboxylic acid is activated with an activatingreagent.

An imine or oxime linker is formed by the reaction of an amine orN-alkoxyamine (or aminooxy) group on one component with an aldehyde orketone group on the second component.

An urea or thiourea linker is prepared by the reaction of an amine groupon one component with an isocyanate or thioisocyanate group on thesecond component.

An amidine linker is prepared by the reaction of an amine group on onecomponent with an imidoester group on the second component.

An amine linker is made by the alkylation reaction of an amine group onone component with an alkylating group such as halide, epoxide, orsulfonate ester group on the second component. Alternatively, an aminelinker can also be made by reductive amination of an amine group on onecomponent with an aldehyde or ketone group on the second component witha suitable reducing reagent such as sodium cyanoborohydride or sodiumtriacetoxyborohydride.

A sulfonamide linker is made by the reaction of an amine group on onecomponent with a sulfonyl halide (such as sulfonyl chloride) group onthe second component.

A sulfone linker is made by Michael addition of a nucleophile to a vinylsulfone. Either the vinyl sulfone or the nucleophile may be on thesurface of the nanocarrier or attached to a component.

The component can also be conjugated to the nanocarrier via non-covalentconjugation methods. For example, a negative charged antigen orimmunosuppressant can be conjugated to a positive charged nanocarrierthrough electrostatic adsorption. A component containing a metal ligandcan also be conjugated to a nanocarrier containing a metal complex via ametal-ligand complex.

In embodiments, the component can be attached to a polymer, for examplepolylactic acid-block-polyethylene glycol, prior to the assembly of thesynthetic nanocarrier or the synthetic nanocarrier can be formed withreactive or activatible groups on its surface. In the latter case, thecomponent may be prepared with a group which is compatible with theattachment chemistry that is presented by the synthetic nanocarriers'surface. In other embodiments, a peptide component can be attached toVLPs or liposomes using a suitable linker. A linker is a compound orreagent that capable of coupling two molecules together. In anembodiment, the linker can be a homobifuntional or heterobifunctionalreagent as described in Hermanson 2008. For example, an VLP or liposomesynthetic nanocarrier containing a carboxylic group on the surface canbe treated with a homobifunctional linker, adipic dihydrazide (ADH), inthe presence of EDC to form the corresponding synthetic nanocarrier withthe ADH linker. The resulting ADH linked synthetic nanocarrier is thenconjugated with a peptide component containing an acid group via theother end of the ADH linker on NC to produce the corresponding VLP orliposome peptide conjugate.

For detailed descriptions of available conjugation methods, seeHermanson G T “Bioconjugate Techniques”, 2nd Edition Published byAcademic Press, Inc., 2008. In addition to covalent attachment thecomponent can be coupled by adsorption to a pre-formed syntheticnanocarrier or it can be coupled by encapsulation during the formationof the synthetic nanocarrier.

Any immunosuppressant as provided herein can be coupled to the syntheticnanocarrier. Immunosuppressants include, but are not limited to,statins; mTOR inhibitors, such as rapamycin or a rapamycin analog; TGF-βsignaling agents; TGF-β receptor agonists; histone deacetylase (HDAC)inhibitors; corticosteroids; inhibitors of mitochondrial function, suchas rotenone; P38 inhibitors; NF-κβ inhibitors; adenosine receptoragonists; prostaglandin E2 agonists; phosphodiesterase inhibitors, suchas phosphodiesterase 4 inhibitor; proteasome inhibitors; kinaseinhibitors; G-protein coupled receptor agonists; G-protein coupledreceptor antagonists; glucocorticoids; retinoids; cytokine inhibitors;cytokine receptor inhibitors; cytokine receptor activators; peroxisomeproliferator-activated receptor antagonists; peroxisomeproliferator-activated receptor agonists; histone deacetylaseinhibitors; calcineurin inhibitors; phosphatase inhibitors and oxidizedATPs. Immunosuppressants also include IDO, vitamin D3, cyclosporine A,aryl hydrocarbon receptor inhibitors, resveratrol, azathiopurine,6-mercaptopurine, aspirin, niflumic acid, estriol, tripolide,interleukins (e.g., IL-1, IL-10), cyclosporine A, siRNAs targetingcytokines or cytokine receptors and the like.

Examples of statins include atorvastatin (LIPITOR®, TORVAST®),cerivastatin, fluvastatin (LESCOL®, LESCOL® XL), lovastatin (MEVACOR®,ALTOCOR®, ALTOPREV®), mevastatin (COMPACTIN®), pitavastatin (LIVALO®,PIAVA®), rosuvastatin (PRAVACHOL®, SELEKTINE®, LIPOSTAT®), rosuvastatin(CRESTOR®), and simvastatin (ZOCOR®, LIPEX®).

Examples of mTOR inhibitors include rapamycin and analogs thereof (e.g.,CCL-779, RAD001, AP23573, C20-methallylrapamycin (C20-Marap),C16-(S)-butylsulfonamidorapamycin (C16-BSrap),C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry &Biology 2006, 13:99-107)), AZD8055, BEZ235 (NVP-BEZ235), chrysophanicacid (chrysophanol), deforolimus (MK-8669), everolimus (RAD0001),KU-0063794, PI-103, PP242, temsirolimus, and WYE-354 (available fromSelleck, Houston, Tex., USA).

Examples of TGF-β signaling agents include TGF-β ligands (e.g., activinA, GDF1, GDF11, bone morphogenic proteins, nodal, TGF-βs) and theirreceptors (e.g., ACVR1B, ACVR1C, ACVR2A, ACVR2B, BMPR2, BMPR1A, BMPR1B,TGFβRI, TGFβRII), R-SMADS/co-SMADS (e.g., SMAD1, SMAD2, SMAD3, SMAD4,SMADS, SMAD8), and ligand inhibitors (e.g, follistatin, noggin, chordin,DAN, lefty, LTBP1, THBS1, Decorin).

Examples of inhibitors of mitochondrial function include atractyloside(dipotassium salt), bongkrekic acid (triammonium salt), carbonyl cyanidem-chlorophenylhydrazone, carboxyatractyloside (e.g., from Atractylisgummifera), CGP-37157, (−)-Deguelin (e.g., from Mundulea sericea), F16,hexokinase II VDAC binding domain peptide, oligomycin, rotenone, Ru360,SFK1, and valinomycin (e.g., from Streptomyces fulvissimus)(EMD4Biosciences, USA).

Examples of P38 inhibitors include SB-203580(4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole),SB-239063(trans-1-(4hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxy-pyrimidin-4-yl)imidazole), SB-220025(5-(2amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole)),and ARRY-797.

Examples of NF (e.g., NK-κβ) inhibitors include IFRD1,2-(1,8-naphthyridin-2-yl)-Phenol, 5-aminosalicylic acid, BAY 11-7082,BAY 11-7085, CAPE (Caffeic Acid Phenethylester), diethylmaleate, IKK-2Inhibitor IV, IMD 0354, lactacystin, MG-132 [Z-Leu-Leu-Leu-CHO], NFκBActivation Inhibitor III, NF-κB Activation Inhibitor II, JSH-23,parthenolide, Phenylarsine Oxide (PAO), PPM-18,pyrrolidinedithiocarbamic acid ammonium salt, QNZ, RO 106-9920,rocaglamide, rocaglamide AL, rocaglamide C, rocaglamide I, rocaglamideJ, rocaglaol, (R)-MG-132, sodium salicylate, triptolide (PG490),wedelolactone.

Examples of adenosine receptor agonists include CGS-21680 and ATL-146e.

Examples of prostaglandin E2 agonists include E-Prostanoid 2 andE-Prostanoid 4.

Examples of phosphodiesterase inhibitors (non-selective and selectiveinhibitors) include caffeine, aminophylline, IBMX(3-isobutyl-1-methylxanthine), paraxanthine, pentoxifylline,theobromine, theophylline, methylated xanthines, vinpocetine, EHNA(erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximone (PERFAN™),milrinone, levosimendon, mesembrine, ibudilast, piclamilast, luteolin,drotaverine, roflumilast (DAXAS™, DALIRESP™), sildenafil (REVATION®,VIAGRA®), tadalafil (ADCIRCA®, CIALIS®), vardenafil (LEVITRA®, STAXYN®),udenafil, avanafil, icariin, 4-methylpiperazine, and pyrazolopyrimidin-7-1.

Examples of proteasome inhibitors include bortezomib, disulfiram,epigallocatechin-3-gallate, and salinosporamide A.

Examples of kinase inhibitors include bevacizumab, BIBW 2992, cetuximab(ERBITUX®), imatinib (GLEEVEC®), trastuzumab (HERCEPTIN®), gefitinib(IRESSA®), ranibizumab (LUCENTIS®), pegaptanib, sorafenib, dasatinib,sunitinib, erlotinib, nilotinib, lapatinib, panitumumab, vandetanib,E7080, pazopanib, mubritinib.

Examples of glucocorticoids include hydrocortisone (cortisol), cortisoneacetate, prednisone, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone acetate,deoxycorticosterone acetate (DOCA), and aldosterone.

Examples of retinoids include retinol, retinal, tretinoin (retinoicacid, RETIN-A®), isotretinoin (ACCUTANE®, AMNESTEEM®, CLARAVIS,SOTRET®), alitretinoin (PANRETIN®), etretinate (TEGISON) and itsmetabolite acitretin (SORIATANE®), tazarotene (TAZORAC®, AVAGE®,ZORAC®), bexarotene (TARGRETIN®), and adapalene (DIFFERIN®).

Examples of cytokine inhibitors include IL1ra, IL1 receptor antagonist,IGFBP, TNF-BF, uromodulin, Alpha-2-Macroglobulin, Cyclosporin A,Pentamidine, and Pentoxifylline (PENTOPAK®, PENTOXIL®, TRENTAL®).

Examples of peroxisome proliferator-activated receptor antagonistsinclude GW9662, PPARγ antagonist III, G335, T0070907 (EMD4Biosciences,USA).

Examples of peroxisome proliferator-activated receptor agonists includepioglitazone, ciglitazone, clofibrate, GW1929, GW7647, L-165,041, LY171883, PPARγ activator, Fmoc-Leu, troglitazone, and WY-14643(EMD4Biosciences, USA).

Examples of histone deacetylase inhibitors include hydroxamic acids (orhydroxamates) such as trichostatin A, cyclic tetrapeptides (such astrapoxin B) and depsipeptides, benzamides, electrophilic ketones,aliphatic acid compounds such as phenylbutyrate and valproic acid,hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101), LAQ824,and panobinostat (LBH589), benzamides such as entinostat (MS-275),CI994, and mocetinostat (MGCD0103), nicotinamide, derivatives of NAD,dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.

Examples of calcineurin inhibitors include cyclosporine, pimecrolimus,voclosporin, and tacrolimus.

Examples of phosphatase inhibitors include BN82002 hydrochloride,CP-91149, calyculin A, cantharidic acid, cantharidin, cypermethrin,ethyl-3,4-dephostatin, fostriecin sodium salt, MAZ51,methyl-3,4-dephostatin, NSC 95397, norcantharidin, okadaic acid ammoniumsalt from prorocentrum concavum, okadaic acid, okadaic acid potassiumsalt, okadaic acid sodium salt, phenylarsine oxide, various phosphataseinhibitor cocktails, protein phosphatase 1C, protein phosphatase 2Ainhibitor protein, protein phosphatase 2A1, protein phosphatase 2A2,sodium orthovanadate.

In some embodiments, the therapeutic protein APC presentable antigens asdescribed herein are also coupled to synthetic nanocarriers. In someembodiments, the therapeutic protein APC presentable antigens arecoupled to the same or different synthetic nanocarriers as to which theimmunosuppressants are coupled. In other embodiments, the therapeuticprotein APC presentable antigens are not coupled to any syntheticnanocarriers. Therapeutic protein APC presentable antigens include anyof the therapeutic proteins, or fragments or derivatives thereof,provided herein.

Therapeutic proteins include, but are not limited to, infusibletherapeutic proteins, enzymes, enzyme cofactors, hormones, bloodclotting factors, cytokines and interferons, growth factors, monoclonalantibodies, and polyclonal antibodies (e.g., that are administered to asubject as a replacement therapy), and proteins associated with Pompe'sdisease (e.g., alglucosidase alfa, rhGAA (e.g., Myozyme and Lumizyme(Genzyme)). Therapeutic proteins also include proteins involved in theblood coagulation cascade. Therapeutic proteins include, but are notlimited to, Factor VIII, Factor VII, Factor IX, Factor V, von WillebrandFactor, von Heldebrant Factor, tissue plasminogen activator, insulin,growth hormone, erythropoietin alfa, VEGF, thrombopoietin, lysozyme,antithrombin and the like. Therapeutic proteins also include adipokines,such as leptin and adiponectin. Other examples of therapeutic proteinsare as described below and elsewhere herein. Also included are fragmentsor derivatives of any of the therapeutic proteins provided as theantigen.

Examples of therapeutic proteins used in enzyme replacement therapy ofsubjects having a lysosomal storage disorder include, but are notlimited to, imiglucerase for the treatment of Gaucher's disease (e.g.,CEREZYME™), a-galactosidase A (a-gal A) for the treatment of Fabrydisease (e.g., agalsidase beta, FABRYZYME™), acid a-glucosidase (GAA)for the treatment of Pompe disease (e.g., alglucosidase alfa, LUMIZYME™,MYOZYME™), arylsulfatase B for the treatment of Mucopolysaccharidoses(e.g., laronidase, ALDURAZYME™, idursulfase, ELAPRASE™, arylsulfatase B,NAGLAZYME™).

Examples of enzymes include oxidoreductases, transferases, hydrolases,lyases, isomerases, and ligases.

Examples of hormones include Melatonin (N-acetyl-5-methoxytryptamine),Serotonin, Thyroxine (or tetraiodothyronine) (a thyroid hormone),Triiodothyronine (a thyroid hormone), Epinephrine (or adrenaline),Norepinephrine (or noradrenaline), Dopamine (or prolactin inhibitinghormone), Antimullerian hormone (or mullerian inhibiting factor orhormone), Adiponectin, Adrenocorticotropic hormone (or corticotropin),Angiotensinogen and angiotensin, Antidiuretic hormone (or vasopressin,arginine vasopressin), Atrial-natriuretic peptide (or atriopeptin),Calcitonin, Cholecystokinin, Corticotropin-releasing hormone,Erythropoietin, Follicle-stimulating hormone, Gastrin, Ghrelin,Glucagon, Glucagon-like peptide (GLP-1), GIP, Gonadotropin-releasinghormone, Growth hormone-releasing hormone, Human chorionic gonadotropin,Human placental lactogen, Growth hormone, Inhibin, Insulin, Insulin-likegrowth factor (or somatomedin), Leptin, Luteinizing hormone, Melanocytestimulating hormone, Orexin, Oxytocin, Parathyroid hormone, Prolactin,Relaxin, Secretin, Somatostatin, Thrombopoietin, Thyroid-stimulatinghormone (or thyrotropin), Thyrotropin-releasing hormone, Cortisol,Aldosterone, Testosterone, Dehydroepiandrosterone, Androstenedione,Dihydrotestosterone, Estradiol, Estrone, Estriol, Progesterone,Calcitriol (1,25-dihydroxyvitamin D3), Calcidiol (25-hydroxyvitamin D3),Prostaglandins, Leukotrienes, Prostacyclin, Thromboxane, Prolactinreleasing hormone, Lipotropin, Brain natriuretic peptide, NeuropeptideY, Histamine, Endothelin, Pancreatic polypeptide, Renin, and Enkephalin.

Examples of blood and blood coagulation factors include Factor I(fibrinogen), Factor II (prothrombin), tissue factor, Factor V(proaccelerin, labile factor), Factor VII (stable factor, proconvertin),Factor VIII (antihemophilic globulin), Factor IX (Christmas factor orplasma thromboplastin component), Factor X (Stuart-Prower factor),Factor Xa, Factor XI, Factor XII (Hageman factor), Factor XIII(fibrin-stabilizing factor), von Willebrand factor, prekallikrein(Fletcher factor), high-molecular weight kininogen (HMWK) (Fitzgeraldfactor), fibronectin, fibrin, thrombin, antithrombin III, heparincofactor II, protein C, protein S, protein Z, protein Z-related proteaseinhibitot (ZPI), plasminogen, alpha 2-antiplasmin, tissue plasminogenactivator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI1),plasminogen activator inhibitor-2 (PAI2), cancer procoagulant, andepoetin alfa (Epogen, Procrit).

Examples of cytokines include lymphokines, interleukins, and chemokines,type 1 cytokines, such as IFN-γ, TGF-β, and type 2 cytokines, such asIL-4, IL-10, and IL-13.

Examples of growth factors include Adrenomedullin (AM), Angiopoietin(Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs),Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF),Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cellline-derived neurotrophic factor (GDNF), Granulocyte colony-stimulatingfactor (G-CSF), Granulocyte macrophage colony-stimulating factor(GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growthfactor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growthfactor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nervegrowth factor (NGF) and other neurotrophins, Platelet-derived growthfactor (PDGF), Thrombopoietin (TPO), Transforming growth factor alpha(TGF-α), Transforming growth factor beta(TGF-β),Tumour_necrosis_factor-alpha (TNF-α), Vascular endothelial growth factor(VEGF), Wnt Signaling Pathway, placental growth factor (P1GF), [(FoetalBovine Somatotrophin)] (FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, andIL-7.

Examples of monoclonal antibodies include Abagovomab, Abciximab,Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD,Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Anrukinzumab,Anti-thymocyte globin, Apolizumab, Arcitumomab, Aselizumab, Atlizumab(tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab, Bavituximab,Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab,Bevacizumab, Biciromab, Bivatuzumab mertansine, Blinatumomab,Brentuximab vedotin, Briakinumab, Canakinumab, Cantuzumab mertansine,Capromab pendetide, Catumaxomab, Cedelizumab, Certolizumab pegol,Cetuximab, Citatuzumab bogatox, Cixutumumab, Clenoliximab, Clivatuzumabtetraxetan, Conatumumab, Dacetuzumab, Daclizumab, Daratumumab,Denosumab, Detumomab, Dorlimomab aritox, Dorlixizumab, Ecromeximab,Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elotuzumab,Elsilimomab, Enlimomab pegol, Epitumomab cituxetan, Epratuzumab,Erlizumab, Ertumaxomab, Etaracizumab, Exbivirumab, Fanolesomab,Faralimomab, Farletuzumab, Felvizumab, Fezakinumab, Figitumumab,Fontolizumab, Foravirumab, Fresolimumab, Galiximab, Gantenerumab,Gavilimomab, Gemtuzumab ozogamicin, GC1008, Girentuximab, Glembatumumabvedotin, Golimumab, Gomiliximab, Ibalizumab, Ibritumomab tiuxetan,Igovomab, Imciromab, Infliximab, Intetumumab, Inolimomab, Inotuzumabozogamicin, Ipilimumab, Iratumumab, Keliximab, Labetuzumab,Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab,Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab,Mapatumumab, Maslimomab, Matuzumab, Mepolizumab, Metelimumab,Milatuzumab, Minretumomab, Mitumomab, Morolimumab, Motavizumab,Muromonab-CD3, Nacolomab tafenatox, Naptumomab estafenatox, Natalizumab,Nebacumab, Necitumumab, Nerelimomab, Nimotuzumab, Nofetumomab merpentan,Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Omalizumab, Oportuzumabmonatox, Oregovomab, Otelixizumab, Pagibaximab, Palivizumab,Panitumumab, Panobacumab, Pascolizumab, Pemtumomab, Pertuzumab,Pexelizumab, Pintumomab, Priliximab, Pritumumab, Rafivirumab,Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab Reslizumab,Rilotumumab, Rituximab, Robatumumab, Rontalizumab, Rovelizumab,Ruplizumab, Satumomab pendetide, Sevirumab, Sibrotuzumab, Sifalimumab,Siltuximab, Siplizumab, Solanezumab, Sonepcizumab, Sontuzumab,Stamulumab, Sulesomab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab,Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomab aritox,Tenatumomab, Teneliximab, Teplizumab, Ticilimumab (tremelimumab),Tigatuzumab, Tocilizumab (atlizumab), Toralizumab, Tositumomab,Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab,Urtoxazumab, Ustekinumab, Vapaliximab, Vedolizumab, Veltuzumab,Vepalimomab, Visilizumab, Volociximab, Votumumab, Zalutumumab,Zanolimumab, Ziralimumab, and Zolimomab aritox.

Examples of infusion therapy or injectable therapeutic proteins include,for example, Tocilizumab (Roche/Actemra®), alpha-1 antitryp sin(Kamada/AAT), Hematide® (Affymax and Takeda, synthetic peptide),albinterferon alfa-2b (Novartis/Zalbin™), Rhucin® (Pharming Group, C1inhibitor replacement therapy), tesamorelin (Theratechnologies/Egrifta,synthetic growth hormone-releasing factor), ocrelizumab (Genentech,Roche and Biogen), belimumab (GlaxoSmithKline/Benlysta®), pegloticase(Savient Pharmaceuticals/Krystexxa™), taliglucerase alfa(Protalix/Uplyso), agalsidase alfa (Shire/Replagal®), velaglucerase alfa(Shire).

Additional therapeutic proteins useful in accordance to aspects of thisinvention will be apparent to those of skill in the art, and theinvention is not limited in this respect.

In some embodiments, a component, such as an antigen orimmunosuppressant, may be isolated. Isolated refers to the element beingseparated from its native environment and present in sufficientquantities to permit its identification or use. This means, for example,the element may be (i) selectively produced by expression cloning or(ii) purified as by chromatography or electrophoresis. Isolated elementsmay be, but need not be, substantially pure. Because an isolated elementmay be admixed with a pharmaceutically acceptable excipient in apharmaceutical preparation, the element may comprise only a smallpercentage by weight of the preparation. The element is nonethelessisolated in that it has been separated from the substances with which itmay be associated in living systems, i.e., isolated from other lipids orproteins. Any of the elements provided herein may be isolated andincluded in the compositions in isolated form.

D. METHODS OF MAKING AND USING THE INVENTIVE COMPOSITIONS AND RELATEDMETHODS

Synthetic nanocarriers may be prepared using a wide variety of methodsknown in the art. For example, synthetic nanocarriers can be formed bymethods as nanoprecipitation, flow focusing using fluidic channels,spray drying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanomaterials have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843). Additional methods havebeen described in the literature (see, e.g., Doubrow, Ed.,“Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press,Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13;Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz etal., 1988, J. Appl. Polymer Sci., 35:755; U.S. Pat. Nos. 5,578,325 and6,007,845; P. Paolicelli et al., “Surface-modified PLGA-basedNanoparticles that can Efficiently Associate and Deliver Virus-likeParticles” Nanomedicine. 5(6):843-853 (2010)).

Various materials may be encapsulated into synthetic nanocarriers asdesirable using a variety of methods including but not limited to C.Astete et al., “Synthesis and characterization of PLGA nanoparticles” J.Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K.Avgoustakis “Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide)Nanoparticles: Preparation, Properties and Possible Applications in DrugDelivery” Current Drug Delivery 1:321-333 (2004); C. Reis et al.,“Nanoencapsulation I. Methods for preparation of drug-loaded polymericnanoparticles” Nanomedicine 2:8-21 (2006); P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010). Other methods suitable for encapsulating materials intosynthetic nanocarriers may be used, including without limitation methodsdisclosed in U.S. Pat. No. 6,632,671 to Unger Oct. 14, 2003.

In certain embodiments, synthetic nanocarriers are prepared by ananoprecipitation process or spray drying. Conditions used in preparingsynthetic nanocarriers may be altered to yield particles of a desiredsize or property (e.g., hydrophobicity, hydrophilicity, externalmorphology, “stickiness,” shape, etc.). The method of preparing thesynthetic nanocarriers and the conditions (e.g., solvent, temperature,concentration, air flow rate, etc.) used may depend on the materials tobe coupled to the synthetic nanocarriers and/or the composition of thepolymer matrix.

If particles prepared by any of the above methods have a size rangeoutside of the desired range, particles can be sized, for example, usinga sieve.

Elements (i.e., components) of the inventive synthetic nanocarriers(such as moieties of which an immunofeature surface is comprised,targeting moieties, polymeric matrices, antigens, immunosuppressants andthe like) may be coupled to the overall synthetic nanocarrier, e.g., byone or more covalent bonds, or may be coupled by means of one or morelinkers. Additional methods of functionalizing synthetic nanocarriersmay be adapted from Published US Patent Application 2006/0002852 toSaltzman et al., Published US Patent Application 2009/0028910 toDeSimone et al., or Published International Patent ApplicationWO/2008/127532 A1 to Murthy et al.

Alternatively or additionally, synthetic nanocarriers can be coupled tocomponents directly or indirectly via non-covalent interactions. Innon-covalent embodiments, the non-covalent coupling is mediated bynon-covalent interactions including but not limited to chargeinteractions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. Such couplingsmay be arranged to be on an external surface or an internal surface ofan inventive synthetic nanocarrier. In embodiments, encapsulation and/orabsorption is a form of coupling. In embodiments, the inventivesynthetic nanocarriers can be combined with an antigen by admixing inthe same vehicle or delivery system.

Populations of synthetic nanocarriers may be combined to formpharmaceutical dosage forms according to the present invention usingtraditional pharmaceutical mixing methods. These include liquid-liquidmixing in which two or more suspensions, each containing one or moresubsets of nanocarriers, are directly combined or are brought togethervia one or more vessels containing diluent. As synthetic nanocarriersmay also be produced or stored in a powder form, dry powder-powdermixing could be performed as could the re-suspension of two or morepowders in a common media. Depending on the properties of thenanocarriers and their interaction potentials, there may be advantagesconferred to one or another route of mixing.

Typical inventive compositions that comprise synthetic nanocarriers maycomprise inorganic or organic buffers (e.g., sodium or potassium saltsof phosphate, carbonate, acetate, or citrate) and pH adjustment agents(e.g., hydrochloric acid, sodium or potassium hydroxide, salts ofcitrate or acetate, amino acids and their salts) antioxidants (e.g.,ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20,polysorbate 80, polyoxyethylene 9-10 nonyl phenol, sodiumdesoxycholate), solution and/or cryo/lyo stabilizers (e.g., sucrose,lactose, mannitol, trehalose), osmotic adjustment agents (e.g., salts orsugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin),antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g.,thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers andviscosity-adjustment agents (e.g., polyvinylpyrrolidone, poloxamer 488,carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethyleneglycol, ethanol).

Compositions according to the invention comprise inventive syntheticnanocarriers in combination with pharmaceutically acceptable excipients.The compositions may be made using conventional pharmaceuticalmanufacturing and compounding techniques to arrive at useful dosageforms. Techniques suitable for use in practicing the present inventionmay be found in Handbook of Industrial Mixing: Science and Practice,Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta,2004 John Wiley & Sons, Inc.; and Pharmaceutics: The Science of DosageForm Design, 2nd Ed. Edited by M. E. Auten, 2001, Churchill Livingstone.In an embodiment, inventive synthetic nanocarriers are suspended insterile saline solution for injection together with a preservative.

It is to be understood that the compositions of the invention can bemade in any suitable manner, and the invention is in no way limited tocompositions that can be produced using the methods described herein.Selection of an appropriate method may require attention to theproperties of the particular moieties being associated.

In some embodiments, inventive synthetic nanocarriers are manufacturedunder sterile conditions or are terminally sterilized. This can ensurethat resulting compositions are sterile and non-infectious, thusimproving safety when compared to non-sterile compositions. Thisprovides a valuable safety measure, especially when subjects receivingsynthetic nanocarriers have immune defects, are suffering frominfection, and/or are susceptible to infection. In some embodiments,inventive synthetic nanocarriers may be lyophilized and stored insuspension or as lyophilized powder depending on the formulationstrategy for extended periods without losing activity.

The compositions of the invention can be administered by a variety ofroutes, including but not limited to subcutaneous, intranasal, oral,intravenous, intraperitoneal, intramuscular, transmucosal, transmucosal,sublingual, rectal, ophthalmic, pulmonary, intradermal, transdermal,transcutaneous or intradermal or by a combination of these routes.Routes of administration also include administration by inhalation orpulmonary aerosol. Techniques for preparing aerosol delivery systems arewell known to those of skill in the art (see, for example, Sciarra andCutie, “Aerosols,” in Remington's Pharmaceutical Sciences, 18th edition,1990, pp. 1694-1712; incorporated by reference).

The therapeutic proteins provided as a cell-based therapy of theinvention may be administered by parenteral, intraarterial, intranasalor intravenous administration or by injection to lymph nodes or anteriorchamber of the eye or by local administration to an organ or tissue ofinterest. The administration may be by subcutaneous, intrathecal,intraventricular, intramuscular, intraperitoneal, intracoronary,intrapancreatic, intrahepatic or bronchial injection.

The compositions of the invention can be administered in effectiveamounts, such as the effective amounts described elsewhere herein. Dosesof dosage forms contain varying amounts of populations of syntheticnanocarriers and/or varying amounts of immunosuppressants and/orantigens, according to the invention. The amount of syntheticnanocarriers and/or immunosuppressants and/or antigens present in theinventive dosage forms can be varied according to the nature of theantigens and/or immunosuppressants, the therapeutic benefit to beaccomplished, and other such parameters. In embodiments, dose rangingstudies can be conducted to establish optimal therapeutic amount of thepopulation of synthetic nanocarriers and the amount ofimmunosuppressants and/or antigens to be present in the dosage form. Inembodiments, the synthetic nanocarriers and/or the immunosuppressantsand/or antigens are present in the dosage form in an amount effective togenerate a tolerogenic immune response to the antigens uponadministration to a subject. It may be possible to determine amounts ofthe immunosuppressants and/or antigens effective to generate atolerogenic immune response using conventional dose ranging studies andtechniques in subjects. Inventive dosage forms may be administered at avariety of frequencies. In a preferred embodiment, at least oneadministration of the dosage form is sufficient to generate apharmacologically relevant response. In more preferred embodiments, atleast two administrations, at least three administrations, or at leastfour administrations, of the dosage form are utilized to ensure apharmacologically relevant response.

Prophylactic administration of the inventive compositions can beinitiated prior to the onset of disease, disorder or condition ortherapeutic administration can be initiated after a disorder, disorderor condition is established.

In some embodiments, administration of synthetic nanocarriers isundertaken e.g., prior to administration of a therapeutic protein. Inexemplary embodiments, synthetic nanocarriers are administered at one ormore times including, but not limited to, 30, 25, 20, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 days prior to administration ofa therapeutic protein. In addition or alternatively, syntheticnanocarriers can be administered to a subject following therapeuticprotein administration. In exemplary embodiments, synthetic nanocarriersare administered at one or more times including, but not limited to, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, etc. daysfollowing administration of a therapeutic protein.

In some embodiments, a maintenance dose (e.g., of a syntheticnanocarrier composition provided herein) is administered to a subjectafter an initial administration has resulted in a tolerogenic responsein the subject, for example, to maintain the tolerogenic effect achievedafter the initial dose, to prevent an undesired immune reaction in thesubject, or to prevent the subject becoming a subject at risk ofexperiencing an undesired immune response or an undesired level of animmune response. In some embodiments, the maintenance dose is the samedose as the initial dose the subject received. In some embodiments, themaintenance dose is a lower dose than the initial dose. For example, insome embodiments, the maintenance dose is about 3/4, about 2/3, about1/2, about 1/3, about 1/4, about 1/8, about 1/10, about 1/20, about1/25, about 1/50, about 1/100, about 1/1,000, about 1/10,000, about1/100,000, or about 1/1,000,000 (weight/weight) of the initial dose.

The compositions and methods described herein can be used to induce orenhance a tolerogenic immune response and/or to suppress, modulate,direct or redirect an undesired immune response for the purpose ofimmune suppression. The compositions and methods described herein can beused to for the generation of a tolerogenic immune response in a subjectthat has been, is being or will be administered a therapeutic protein.

EXAMPLES Example 1 Immune Response of Synthetic Nanocarriers withCoupled Rapamycin with and without Ovalbumin Peptide (323-339) Materials

Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T and Bcell epitope of Ovalbumin protein, was purchased from Bachem AmericasInc. (3132 Kashiwa Street, Torrance Calif. 90505; Part #4065609).Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham,Mass. 01702; Product Catalogue # R1017). PLGA with a lactide:glycolideratio of 3:1 and an inherent viscosity of 0.75 dL/g was purchased fromSurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211;Product Code 7525 DLG 7A). Polyvinyl alcohol (85-89% hydrolyzed) waspurchased from EMD Chemicals (Product Number 1.41350.1001).

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13 M hydrochloric acid solution at room temperature.

Solution 2: Rapamycin @ 50 mg/mL in methylene chloride. The solution wasprepared by dissolving rapamycin in pure methylene chloride.

Solution 3: PLGA @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLGA in pure methylene chloride.

Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

Method for Preparing Synthetic Nanocarrier Containing Rapamycin andOvalbumin (323-339)

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining solution 1 (0.2 mL), solution 2 (0.2 mL), and solution 3(1.0 mL) in a small pressure tube and sonicating at 50% amplitude for 40seconds using a Branson Digital Sonifier 250. A secondary emulsion(W1/O1/W2) was then prepared by combining solution 4 (3.0 mL) with theprimary W1/O1 emulsion, vortexing for 10 s, and sonicating at 30%amplitude for 60 seconds using the Branson Digital Sonifier 250.

The W1/O1/W2 emulsion was added to a beaker containing 70 mM pH 8phosphate buffer solution (30 mL) and stirred at room temperature for 2hours to allow the methylene chloride to evaporate and for the syntheticnanocarriers to form. A portion of the synthetic nanocarriers werewashed by transferring the synthetic nanocarrier suspension to acentrifuge tube and centrifuging at 21,000×g and 4° C. for one hour,removing the supernatant, and re-suspending the pellet in phosphatebuffered saline. The washing procedure was repeated, and the pellet wasre-suspended in phosphate buffered saline for a final syntheticnanocarrier dispersion of about 10 mg/mL.

The amounts of peptide and rapamycin in the synthetic nanocarriers weredetermined by HPLC analysis. The total dry-synthetic nanocarrier massper mL of suspension was determined by a gravimetric method.

Method for Synthetic Nanocarrier Containing Rapamycin

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining 0.13 M hydrochloric acid solution (0.2 mL), solution 2 (0.2mL), and solution 3 (1.0 mL) in a small pressure tube and sonicating at50% amplitude for 40 seconds using a Branson Digital Sonifier 250. Asecondary emulsion (W1/O1/W2) was then prepared by combining solution 4(3.0 mL) with the primary W1/O1 emulsion, vortexing for 10 s, andsonicating at 30% amplitude for 60 seconds using the Branson DigitalSonifier 250.

The W1/O1/W2 emulsion was added to a beaker containing 70 mM pH 8phosphate buffer solution (30 mL) and stirred at room temperature for 2hours to allow the methylene chloride to evaporate and for the syntheticnanocarriers to form. A portion of the synthetic nanocarriers werewashed by transferring the synthetic nanocarrier suspension to acentrifuge tube and centrifuging at 21,000×g and 4° C. for one hour,removing the supernatant, and re-suspending the pellet in phosphatebuffered saline. The washing procedure was repeated, and the pellet wasre-suspended in phosphate buffered saline for a final syntheticnanocarrier dispersion of about 10 mg/mL.

The amount of rapamycin in the synthetic nanocarrier was determined byHPLC analysis. The total dry-synthetic nanocarrier mass per mL ofsuspension was determined by a gravimetric method.

Method for Measuring Rapamycin Load

Approximately 3 mg of synthetic nanocarriers were collected andcentrifuged to separate supernatant from synthetic nanocarrier pellet.Acetonitrile was added to the pellet, and the sample was sonicated andcentrifuged to remove any insoluble material. The supernatant and pelletwere injected on RP-HPLC and absorbance was read at 278 nm. The μg foundin the pellet were used to calculate % entrapped (load), μg insupernatant and pellet were used to calculate total μg recovered.

Method for Measuring Ovalbumin (323-339) Load

Approximately 3 mg of synthetic nanocarriers were collected andcentrifuged to separate supernatant from synthetic nanocarrier pellet.Trifluoroethanol was added to the pellet and the sample was sonicated todissolve the polymer, 0.2% trifluoroacetic acid was added and sample wassonicated and then centrifuged to remove any insoluble material. Thesupernatant and pellet were injected on RP-HPLC and absorbance was readat 215 nm. The μg found in the pellet were used to calculate % entrapped(load), μg in supernatant and pellet were used to calculate total μgrecovered.

Antigen-Specific Tolerogenic Dendritic Cells (Tdc) Activity on Treg CellDevelopment

The assay included the use of OTII mice which have a transgenic T-cellreceptor specific for an immune-dominant ovalbumin (323-339). In orderto create antigen-specific tDCs, CD11c+ splenocytes were isolated, andthe ovalbumin (323-339) peptide added in vitro at 1 μg/ml or no antigen.Soluble or nanocarrier-encapsulated rapamycin was then added to the DCsfor 2 hours which were then washed extensively to remove free rapamycinfrom the culture. Purified responder CD4+CD25− cells were isolated fromOTII mice and added to tDC at a 10:1 T to DC ratio. The mixture of tDCand OTII T-cells were then cultured for 4-5 days, and the frequency ofTreg cells (CD4+CD25highFoxP3+) were analyzed by flow cytometry as shownin FIG. 1. Regions were selected based on isotype controls.

Example 2 Mesoporous Silica Nanoparticles with Coupled Ibuprofen(Prophetic)

Mesoporous SiO2 nanoparticle cores are created through a sol-gelprocess. Hexadecyltrimethyl-ammonium bromide (CTAB) (0.5 g) is dissolvedin deionized water (500 mL), and then 2 M aqueous NaOH solution (3.5 mL)is added to the CTAB solution. The solution is stirred for 30 min, andthen Tetraethoxysilane (TEOS) (2.5 mL) is added to the solution. Theresulting gel is stirred for 3 h at a temperature of 80° C. The whiteprecipitate which forms is captured by filtration, followed by washingwith deionized water and drying at room temperature. The remainingsurfactant is then extracted from the particles by suspension in anethanolic solution of HCl overnight. The particles are washed withethanol, centrifuged, and redispersed under ultrasonication. This washprocedure is repeated two additional times.

The SiO2 nanoparticles are then functionalized with amino groups using(3-aminopropyl)-triethoxysilane (APTMS). To do this, the particles aresuspended in ethanol (30 mL), and APTMS (50 μL) is added to thesuspension. The suspension is allowed to stand at room temperature for 2h and then is boiled for 4 h, keeping the volume constant byperiodically adding ethanol. Remaining reactants are removed by fivecycles of washing by centrifugation and redispersing in pure ethanol.

In a separate reaction, 1-4 nm diameter gold seeds are created. Allwater used in this reaction is first deionized and then distilled fromglass. Water (45.5 mL) is added to a 100 mL round-bottom flask. Whilestirring, 0.2 M aqueous NaOH (1.5 mL) is added, followed by a 1% aqueoussolution of tetrakis(hydroxymethyl)phosphonium chloride (THPC) (1.0 mL).Two minutes after the addition of THPC solution, a 10 mg/mL aqueoussolution of chloroauric acid (2 mL), which has been aged at least 15min, is added. The gold seeds are purified through dialysis againstwater.

To form the core-shell nanocarriers, the amino-functionalized SiO2nanoparticles formed above are first mixed with the gold seeds for 2 hat room temperature. The gold-decorated SiO2 particles are collectedthrough centrifugation and mixed with an aqueous solution of chloroauricacid and potassium bicarbonate to form the gold shell. The particles arethen washed by centrifugation and redispersed in water. Ibuprofen isloaded by suspending the particles in a solution of sodium ibuprofen (1mg/L) for 72 h. Free ibuprofen is then washed from the particles bycentrifugation and redispersing in water.

Example 3 Liposomes Containing Cyclosporine A (Prophetic)

The liposomes are formed using thin film hydration.1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 μmol),cholesterol (32 μmol), and cyclosporin A (6.4 μmol) are dissolved inpure chloroform (3 mL). This lipid solution is added to a 50 mLround-bottom flask, and the solvent is evaporated on a rotary evaporatorat a temperature of 60° C. The flask is then flushed with nitrogen gasto remove remaining solvent. Phosphate buffered saline (2 mL) and fiveglass beads are added to the flask, and the lipid film is hydrated byshaking at 60° C. for 1 h to form a suspension. The suspension istransferred to a small pressure tube and sonicated at 60° C. for fourcycles of 30 s pulses with a 30 s delay between each pulse. Thesuspension is then left undisturbed at room temperature for 2 h to allowfor complete hydration. The liposomes are washed by centrifugationfollowed by resuspension in fresh phosphate buffered saline.

Example 4 Polymeric Nanocarrier Containing Polymer-Rapamycin Conjugate(Prophetic)

Preparation of PLGA-Rapamycin Conjugate:

PLGA polymer with acid end group (7525 DLG1A, acid number 0.46 mmol/g,Lakeshore Biomaterials; 5 g, 2.3 mmol, 1.0 eq) is dissolved in 30 mL ofdichloromethane (DCM). N,N-Dicyclohexylcarbodimide (1.2 eq, 2.8 mmol,0.57 g) is added followed by rapamycin (1.0 eq, 2.3 mmol, 2.1 g) and4-dimethylaminopyridine (DMAP) (2.0 eq, 4.6 mmol, 0.56 g). The mixtureis stirred at rt for 2 days. The mixture is then filtered to removeinsoluble dicyclohexylurea. The filtrate is concentrated to ca. 10 mL involume and added to 100 mL of isopropyl alcohol (IPA) to precipitate outthe PLGA-rapamycin conjugate. The IPA layer is removed and the polymeris then washed with 50 mL of IPA and 50 mL of methyl t-butyl ether(MTBE). The polymer is then dried under vacuum at 35 C for 2 days togive PLGA-rapamycin as a white solid (ca. 6.5 g).

Preparation of nanocarrier containing PLGA-rapamycin conjugate andovalbumin peptide (323-339):

Nanocarrier containing PLGA-rapamycin is prepared according to theprocedure described in Example 1 as follows:

Solutions for nanocarrier formation are prepared as follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute hydrochloricacid aqueous solution. The solution is prepared by dissolving ovalbuminpeptide in 0.13 M hydrochloric acid solution at room temperature.Solution 2: PLGA-rapamycin @ 100 mg/mL in methylene chloride. Thesolution is prepared by dissolving PLGA-rapamycin in pure methylenechloride. Solution 3: PLA-PEG @ 100 mg/mL in methylene chloride. Thesolution is prepared by dissolving PLA-PEG in pure methylene chloride.Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

A primary water-in-oil emulsion is prepared first. W1/O1 is prepared bycombining solution 1 (0.2 mL), solution 2 (0.75 mL), and solution 3(0.25 mL) in a small pressure tube and sonicating at 50% amplitude for40 seconds using a Branson Digital Sonifier 250. A secondary emulsion(W1/O1/W2) is then prepared by combining solution 4 (3.0 mL) with theprimary W1/O1 emulsion, vortexing for 10 s, and sonicating at 30%amplitude for 60 seconds using the Branson Digital Sonifier 250. TheW1/O1/W2 emulsion is added to a beaker containing 70 mM pH 8 phosphatebuffer solution (30 mL) and stirred at room temperature for 2 hours toallow the methylene chloride to evaporate and for the nanocarriers toform. A portion of the nanocarriers is washed by transferring thenanocarrier suspension to a centrifuge tube and centrifuging at 75,600×gand 4° C. for 35 min, removing the supernatant, and re-suspending thepellet in phosphate buffered saline. The washing procedure is repeated,and the pellet is re-suspended in phosphate buffered saline for a finalnanocarrier dispersion of about 10 mg/mL.

Example 5 Preparation of Gold Nanocarriers (AuNCs) Containing Rapamycin(Prophetic)

Preparation of HS-PEG-Rapamycin:

A solution of PEG acid disulfide (1.0 eq), rapamycin (2.0-2.5 eq), DCC(2.5 eq) and DMAP (3.0 eq) in dry DMF is stirred at rt overnight. Theinsoluble dicyclohexylurea is removed by filtration and the filtrate isadded to isopropyl alcohol (IPA) to precipitate out thePEG-disulfide-di-rapamycin ester and washed with IPA and dried. Thepolymer is then treated with tris(2-carboxyethyl)phosphine hydrochloridein DMF to reduce the PEG disulfide to thiol PEG rapamycin ester(HS-PEG-rapamycin). The resulting polymer is recovered by precipitationfrom IPA and dried as previously described and analyzed by H NMR andGPC.

Formation of Gold NCs (AuNCs):

An aq. solution of 500 mL of 1 mM HAuCl4 is heated to reflux for 10 minwith vigorous stirring in a 1 L round-bottom flask equipped with acondenser. A solution of 50 mL of 40 mM of trisodium citrate is thenrapidly added to the stirring solution. The resulting deep wine redsolution is kept at reflux for 25-30 min and the heat is withdrawn andthe solution is cooled to room temperature. The solution is thenfiltered through a 0.8 μm membrane filter to give the AuNCs solution.The AuNCs are characterized using visible spectroscopy and transmissionelectron microscopy. The AuNCs are ca. 20 nm diameter capped by citratewith peak absorption at 520 nm.

AuNCs Conjugate with HS-PEG-Rapamycin:

A solution of 150 μl of HS-PEG-rapamycin (10 μM in 10 mM pH 9.0carbonate buffer) is added to 1 mL of 20 nm diameter citrate-capped goldnanocarriers (1.16 nM) to produce a molar ratio of thiol to gold of2500:1. The mixture is stirred at room temperature under argon for 1hour to allow complete exchange of thiol with citrate on the goldnanocarriers. The AuNCs with PEG-rapamycin on the surface is thenpurified by centrifuge at 12,000 g for 30 minutes. The supernatant isdecanted and the pellet containing AuNC-S-PEG-rapamycin is then pelletwashed with 1×PBS buffer. The purified Gold-PEG-rapamycin nanocarriersare then resuspend in suitable buffer for further analysis andbioassays.

Example 6 Mesoporous Silica-Gold Core-Shell Nanocarriers ContainingOvalbumin (Prophetic)

Mesoporous SiO2 nanoparticle cores are created through a sol-gelprocess. Hexadecyltrimethyl-ammonium bromide (CTAB) (0.5 g) is dissolvedin deionized water (500 mL), and then 2 M aqueous NaOH solution (3.5 mL)is added to the CTAB solution. The solution is stirred for 30 min, andthen Tetraethoxysilane (TEOS) (2.5 mL) is added to the solution. Theresulting gel is stirred for 3 h at a temperature of 80° C. The whiteprecipitate which forms is captured by filtration, followed by washingwith deionized water and drying at room temperature. The remainingsurfactant is then extracted from the particles by suspension in anethanolic solution of HCl overnight. The particles are washed withethanol, centrifuged, and redispersed under ultrasonication. This washprocedure is repeated two additional times.

The SiO2 nanoparticles are then functionalized with amino groups using(3-aminopropyl)-triethoxysilane (APTMS). To do this, the particles aresuspended in ethanol (30 mL), and APTMS (50 μL) is added to thesuspension. The suspension is allowed to stand at room temperature for 2h and then is boiled for 4 h, keeping the volume constant byperiodically adding ethanol. Remaining reactants are removed by fivecycles of washing by centrifugation and redispersing in pure ethanol.

In a separate reaction, 1-4 nm diameter gold seeds are created. Allwater used in this reaction is first deionized and then distilled fromglass. Water (45.5 mL) is added to a 100 mL round-bottom flask. Whilestirring, 0.2 M aqueous NaOH (1.5 mL) is added, followed by a 1% aqueoussolution of tetrakis(hydroxymethyl)phosphonium chloride (THPC) (1.0 mL).Two minutes after the addition of THPC solution, a 10 mg/mL aqueoussolution of chloroauric acid (2 mL), which has been aged at least 15min, is added. The gold seeds are purified through dialysis againstwater.

To form the core-shell nanocarriers, the amino-functionalized SiO2nanoparticles formed above are first mixed with the gold seeds for 2 hat room temperature. The gold-decorated SiO2 particles are collectedthrough centrifugation and mixed with an aqueous solution of chloroauricacid and potassium bicarbonate to form the gold shell. The particles arethen washed by centrifugation and redispersed in water. ThiolatedOvalbumin (made by treating Ovalbumin with 2-iminothiolanehydrochloride) is loaded by suspending the particles in a solution ofthiolated Ovalbumin (1 mg/L) for 72 h. The particles is then pelletwashed with 1×PBS (pH 7.4) to remove free protein. The resultingsilica-gold core-shell nanocarriers containing Ovalbumin are thenre-suspended in 1×PBS for further analysis and assays.

Example 7 Liposomes Containing Rapamycin and Ovalbumin (Prophetic)

The liposomes are formed by thin film hydration.1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 μmol),cholesterol (32 μmol), and rapamycin (6.4 μmol) are dissolved in purechloroform (3 mL). This lipid solution is added to a 10 mL glass tubeand the solvent is removed under nitrogen gas stream and desiccated for6 hr. under vacuum. Multilamellar vesicles are obtained by hydration ofthe film with 2.0 ml of 25 mM MOPS buffer pH 8.5, containing excessamount of Ovalbumin. The tube is vortexed until the lipid film is peeledof from the tube surface. To break the multilamellar vesicles intomonolamellar, ten cycles of freezing (liquid nitrogen) and thawing (30°C. water bath) are applied. The sample is then diluted to 1 ml in 25 mMMOPS buffer pH 8.5. Size of the resulting liposome is homogenized byextrusion by passing the sample 10 fold through a 200 nm porepolycarbonate filters. The resulting liposomes are then used for furtheranalysis and bioassays.

Example 8 Polymeric Nanocarriers Composed of Modified Polyamino Acidwith Surface Conjugated Ovalbumin (Prophetic)

Step-1. Preparation of Poly(γ-glutamic acid) (γ-PGA) modified withL-phenylalanine ethyl ester (L-PAE): 4.7 unit mmol of γ-PGA (Mn=300 kD)is dissolved in 0.3 N—NaHCO3 aqueous solution (50 mL). L-PAE (4.7 mmol)and EDC.HCl (4.7 mmol) are added to the solution and stirred for 30 minat 4 C. The solution is then maintained at room temperature withstirring for 24 h. Low-molecular-weight chemicals are removed bydialysis using dialysis membrane with MWCO 50 kD. The resultingγ-PGA-graft-L-PAE is obtained by freeze-drying.

Step-2. Preparation of nanoparticles from γ-PGA-graft-L-PAE polymer:Nanoparticles composed of γ-PGA-graft-L-PAE are prepared by aprecipitation and dialysis method. γ-PGA-graft-L-PAE (20 mg) wasdissolved in 2 ml of DMSO followed by addition of 2 mL of water to forma translucent solution. The solution is then dialyzed against distilledwater using cellulose membrane tubing (50,000 MWCO) to form thenanoparticles and to remove the organic solvents for 72 h at roomtemperature. The distilled water is exchanged at intervals of 12 h. Theresulting nanoparticle solution (10 mg/mL in water) is then used forantigen conjugation.

Step-3. Ovalbumin conjugation to γ-PGA nanoparticles: Surface carboxylicacid groups of the γ-PGA nanoparticles (10 mg/ml) are first activated byEDC and NHS (10 mg/mL each in phosphate buffer, pH 5.8) for 2 h atambient temperature. After pellet washing to remove excess EDC/NHS, theactivated nanoparticles are mixed with 1 mL of Ovalbumin (10 mg/ml) inphosphate-buffered saline (PBS, pH 7.4) and the mixture is incubated at4-8 C for 24 h. The resulting Ovalbumin conjugated γ-PGA nanoparticlesare washed twice with PBS and resuspended at 5 mg/mL in PBS for furtheranalysis and bioassays.

Example 9 Erythropoietin (EPO)-Encapsulated γ-PGA Nanoparticles(Prophetic)

To prepare the EPO-encapsulated γ-PGA nanoparticles, 0.25-4 mg of EPO isdissolved in 1 mL of PBS (pH 7.4) and 1 mL of the γ-PGA-graft-L-PAE (10mg/mL in DMSO) is added to the EPO solution. The resulting solution iscentrifuged at 14,000×g for 15 min and repeatedly rinsed with PBS. Theresulting EPO-encapsulated γ-PGA nanoparticles are then resuspended inPBS (5 mg/mL) for further analysis and bioassay.

Example 10 Preparation of Gold Nanocarriers (AuNCs) Containing Ovalbumin(Prophetic)

Step-1. Formation of Gold NCs (AuNCs): An aq. solution of 500 mL of 1 mMHAuCl4 is heated to reflux for 10 min with vigorous stirring in a 1 Lround-bottom flask equipped with a condenser. A solution of 50 mL of 40mM of trisodium citrate is then rapidly added to the stirring solution.The resulting deep wine red solution is kept at reflux for 25-30 min andthe heat is withdrawn and the solution is cooled to room temperature.The solution is then filtered through a 0.8 μm membrane filter to givethe AuNCs solution. The AuNCs are characterized using visiblespectroscopy and transmission electron microscopy. The AuNCs are ca. 20nm diameter capped by citrate with peak absorption at 520 nm.

Step-2. Conjugation of Ovalbumin to AuNCs: A solution of 150 μl ofthiolated Ovalbumin (10 μM in 10 mM pH 9.0 carbonate buffer) is added to1 mL of 20 nm diameter citrate-capped gold nanocarriers (1.16 nM) toproduce a molar ratio of thiol to gold of 2500:1. The mixture is stirredat room temperature under argon for 1 hour to allow complete exchange ofthiol with citrate on the gold nanocarriers. The AuNCs with Ovalbumin onthe surface is then purified by centrifuge at 12,000 g for 30 minutes.The supernatant is decanted and the pellet containing AuNC-Ovalbumin isthen pellet washed with 1×PBS buffer. The purified Gold-Ovalbuminnanocarriers are then resuspend in suitable buffer for further analysisand bioassays.

Example 11 Evaluating Tolerogenic Immune Response to Epoietin Alpha InVivo (Prophetic)

Balb/c mice are immunized with epoietin alpha in incomplete Freundsadjuvant to induce CD4+ T-cell proliferation, the level of which isassessed. Subsequently, a composition of the invention comprising MHCClass II-restricted epitopes of epoietin alpha and an immunosuppressantis administered subcutaneously in a dose-dependent manner. The same miceare then again exposed to the epoietin alpha, and the level of CD4+ Tcell proliferation is again assessed. Changes in the CD4+ T cellpopulation are then monitored with a reduction in CD4+ T cellproliferation upon subsequent challenge with epoietin alpha indicating atolerogenic immune response.

Example 12 Evaluating Tolerogenic Immune Responses with SyntheticNanocarriers Comprising Immunosuppressant and APC Presentable Antigen InVivo Materials and Methods of Synthetic Nanocarrier ProductionNanocarrier 1

Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham,Mass. 01702; Product Catalogue # R1017). PLGA with a lactide:glycolideratio of 3:1 and an inherent viscosity of 0.75 dL/g was purchased fromSurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211;Product Code 7525 DLG 7A). PLA-PEG block co-polymer with a PEG block ofapproximately 5,000 Da and PLA block of approximately 20,000 Da wassynthesized. Polyvinyl alcohol (85-89% hydrolyzed) was purchased fromEMD Chemicals (Product Number 1.41350.1001).

Solutions were Prepared as Follows:

Solution 1: Rapamycin @ 50 mg/mL in methylene chloride. The solution wasprepared by dissolving rapamycin in pure methylene chloride.

Solution 2: PLGA @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLGA in pure methylene chloride.

Solution 3: PLA-PEG @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLA-PEG in pure methylene chloride.

Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

An oil-in-water emulsion was used to prepare the nanocarriers. The O/Wemulsion was prepared by combining solution 1 (0.2 mL), solution 2 (0.75mL), solution 3 (0.25 mL), and solution 4 (3 mL) in a small pressuretube and sonicating at 30% amplitude for 60 seconds using a BransonDigital Sonifier 250. The O/W emulsion was added to a beaker containing70 mM pH 8 phosphate buffer solution (30 mL) and stirred at roomtemperature for 2 hours to allow the methylene chloride to evaporate andfor the nanocarriers to form. A portion of the nanocarriers was washedby transferring the nanocarrier suspension to a centrifuge tube andcentrifuging at 21,000×g and 4° C. for 45 min, removing the supernatant,and re-suspending the pellet in phosphate buffered saline. The washingprocedure was repeated, and the pellet was re-suspended in phosphatebuffered saline for a final nanocarrier dispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountof rapamycin in the nanocarrier was determined by HPLC analysis. Thetotal dry-nanocarrier mass per mL of suspension was determined by agravimetric method.

Effective Diameter Rapamycin Content Nanocarrier ID (nm) (% w/w)Nanocarrier 1 215 9.5

Nanocarrier 2

Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T and Bcell epitope of Ovalbumin protein, was purchased from Bachem AmericasInc. (3132 Kashiwa Street, Torrance Calif. 90505; Part #4065609). PLGAwith a lactide:glycolide ratio of 3:1 and an inherent viscosity of 0.75dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,Birmingham, Ala. 35211; Product Code 7525 DLG 7A). PLA-PEG blockco-polymer with a PEG block of approximately 5,000 Da and PLA block ofapproximately 20,000 Da was synthesized. Polyvinyl alcohol (85-89%hydrolyzed) was purchased from EMD Chemicals (Product Number1.41350.1001).

Solutions were Prepared as Follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13 M hydrochloric acid solution at room temperature.

Solution 2: PLGA @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLGA in pure methylene chloride.

Solution 3: PLA-PEG @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLA-PEG in pure methylene chloride.

Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining solution 1 (0.2 mL), solution 2 (0.75 mL), and solution 3(0.25 mL) in a small pressure tube and sonicating at 50% amplitude for40 seconds using a Branson Digital Sonifier 250. A secondary emulsion(W1/O1/W2) was then prepared by combining solution 4 (3.0 mL) with theprimary W1/O1 emulsion, vortexing for 10 s, and sonicating at 30%amplitude for 60 seconds using the Branson Digital Sonifier 250.

The W1/O1/W2 emulsion was added to a beaker containing 70 mM pH 8phosphate buffer solution (30 mL) and stirred at room temperature for 2hours to allow the methylene chloride to evaporate and for thenanocarriers to form. A portion of the nanocarriers were washed bytransferring the nanocarrier suspension to a centrifuge tube andcentrifuging at 75,600×g and 4° C. for 35 min, removing the supernatant,and re-suspending the pellet in phosphate buffered saline. The washingprocedure was repeated, and the pellet was re-suspended in phosphatebuffered saline for a final nanocarrier dispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountof peptide in the nanocarrier was determined by HPLC analysis. The totaldry-nanocarrier mass per mL of suspension was determined by agravimetric method.

Effective Diameter Peptide Content Nanocarrier ID (nm) (% w/w)Nanocarrier 2 234 2.1

Nanocarrier 3

Simvastatin was purchased from LKT Laboratories, Inc. (2233 UniversityAvenue West, St. Paul, Minn. 55114; Product Catalogue # S3449). PLGAwith a lactide:glycolide ratio of 3:1 and an inherent viscosity of 0.75dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,Birmingham, Ala. 35211; Product Code 7525 DLG 7A). PLA-PEG blockco-polymer with a PEG block of approximately 5,000 Da and PLA block ofapproximately 20,000 Da was synthesized. Polyvinyl alcohol (85-89%hydrolyzed) was purchased from EMD Chemicals (Product Number1.41350.1001).

Solutions were Prepared as Follows:

Solution 1: Simvastatin @ 50 mg/mL in methylene chloride. The solutionwas prepared by dissolving simvastatin in pure methylene chloride.

Solution 2: PLGA @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLGA in pure methylene chloride.

Solution 3: PLA-PEG @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLA-PEG in pure methylene chloride.

Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

An oil-in-water emulsion was used to prepare the nanocarriers. The O/Wemulsion was prepared by combining solution 1 (0.15 mL), solution 2(0.75 mL), solution 3 (0.25 mL), and solution 4 (3 mL) in a smallpressure tube and sonicating at 30% amplitude for 60 seconds using aBranson Digital Sonifier 250. The O/W emulsion was added to a beakercontaining 70 mM pH 8 phosphate buffer solution (30 mL) and stirred atroom temperature for 2 hours to allow the methylene chloride toevaporate and for the nanocarriers to form. A portion of thenanocarriers was washed by transferring the nanocarrier suspension to acentrifuge tube and centrifuging at 75,600×g and 4° C. for 35 min,removing the supernatant, and re-suspending the pellet in phosphatebuffered saline. The washing procedure was repeated, and the pellet wasre-suspended in phosphate buffered saline for a final nanocarrierdispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountof simvastatin in the nanocarrier was determined by HPLC analysis. Thetotal dry-nanocarrier mass per mL of suspension was determined by agravimetric method.

Effective Diameter Simvastatin Content Nanocarrier ID (nm) (% w/w)Nanocarrier 3 196 8.0

Nanocarrier 4

Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T and Bcell epitope of Ovalbumin protein, was purchased from Bachem AmericasInc. (3132 Kashiwa Street, Torrance Calif. 90505; Part #4065609).Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham,Mass. 01702; Product Catalogue # R1017). PLGA with a lactide:glycolideratio of 3:1 and an inherent viscosity of 0.75 dL/g was purchased fromSurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211;Product Code 7525 DLG 7A). PLA-PEG block co-polymer with a PEG block ofapproximately 5,000 Da and PLA block of approximately 20,000 Da wassynthesized. Polyvinyl alcohol (85-89% hydrolyzed) was purchased fromEMD Chemicals (Product Number 1.41350.1001).

Solutions were Prepared as Follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13 M hydrochloric acid solution at room temperature.

Solution 2: Rapamycin @ 50 mg/mL in methylene chloride. The solution wasprepared by dissolving rapamycin in pure methylene chloride.

Solution 3: PLGA @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLGA in pure methylene chloride.

Solution 4: PLA-PEG @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLA-PEG in pure methylene chloride.

Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining solution 1 (0.2 mL), solution 2 (0.2 mL), solution 3 (0.75mL), and solution 4 (0.25 mL) in a small pressure tube and sonicating at50% amplitude for 40 seconds using a Branson Digital Sonifier 250. Asecondary emulsion (W1/O1/W2) was then prepared by combining solution 5(3.0 mL) with the primary W1/O1 emulsion, vortexing for 10 s, andsonicating at 30% amplitude for 60 seconds using the Branson DigitalSonifier 250.

The W1/O1/W2 emulsion was added to a beaker containing 70 mM pH 8phosphate buffer solution (30 mL) and stirred at room temperature for 2hours to allow the methylene chloride to evaporate and for thenanocarriers to form. A portion of the nanocarriers were washed bytransferring the nanocarrier suspension to a centrifuge tube andcentrifuging at 21,000×g and 4° C. for 45 min, removing the supernatant,and re-suspending the pellet in phosphate buffered saline. The washingprocedure was repeated, and the pellet was re-suspended in phosphatebuffered saline for a final nanocarrier dispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountsof peptide and rapamycin in the nanocarrier were determined by HPLCanalysis. The total dry-nanocarrier mass per mL of suspension wasdetermined by a gravimetric method.

Nanocarrier Effective Rapamycin Content Peptide Content ID Diameter (nm)(% w/w) (% w/w) 4 227 9.0 2.5

Nanocarrier 5

Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T and Bcell epitope of Ovalbumin protein, was purchased from Bachem AmericasInc. (3132 Kashiwa Street, Torrance Calif. 90505; Part #4065609).Simvastatin was purchased from LKT Laboratories, Inc. (2233 UniversityAvenue West, St. Paul, Minn. 55114; Product Catalogue # S3449). PLGAwith a lactide:glycolide ratio of 3:1 and an inherent viscosity of 0.75dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,Birmingham, Ala. 35211; Product Code 7525 DLG 7A). PLA-PEG blockco-polymer with a PEG block of approximately 5,000 Da and PLA block ofapproximately 20,000 Da was synthesized. Polyvinyl alcohol (85-89%hydrolyzed) was purchased from EMD Chemicals (Product Number1.41350.1001).

Solutions were Prepared as Follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13 M hydrochloric acid solution at room temperature.

Solution 2: Simvastatin @ 50 mg/mL in methylene chloride. The solutionwas prepared by dissolving simvastatin in pure methylene chloride.

Solution 3: PLGA @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLGA in pure methylene chloride.

Solution 4: PLA-PEG @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLA-PEG in pure methylene chloride.

Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining solution 1 (0.2 mL), solution 2 (0.15 mL), solution 3 (0.75mL), and solution 4 (0.25 mL) in a small pressure tube and sonicating at50% amplitude for 40 seconds using a Branson Digital Sonifier 250. Asecondary emulsion (W1/O1/W2) was then prepared by combining solution 5(3.0 mL) with the primary W1/O1 emulsion, vortexing for 10 s, andsonicating at 30% amplitude for 60 seconds using the Branson DigitalSonifier 250.

The W1/O1/W2 emulsion was added to a beaker containing 70 mM pH 8phosphate buffer solution (30 mL) and stirred at room temperature for 2hours to allow the methylene chloride to evaporate and for thenanocarriers to form. A portion of the nanocarriers were washed bytransferring the nanocarrier suspension to a centrifuge tube andcentrifuging at 75,600×g and 4° C. for 35 min, removing the supernatant,and re-suspending the pellet in phosphate buffered saline. The washingprocedure was repeated, and the pellet was re-suspended in phosphatebuffered saline for a final nanocarrier dispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountsof peptide and simvastatin in the nanocarrier were determined by HPLCanalysis. The total dry-nanocarrier mass per mL of suspension wasdetermined by a gravimetric method.

Effective Simvastatin Content Peptide Content Nanocarrier ID Diameter(nm) (% w/w) (% w/w) Nanocarrier 5 226 2.7 1.9

In Vivo Administration 1

Spleens from B6.Cg-Tg(TcraTcrb)425Cbn/J (OTII) and C57BL/6 (B6) micewere harvested, mechanically dissociated and filtered separately througha 70 μM sieve to yield a single-cell suspension. Purified CD4⁺CD25−cells were then extracted in a 2-step process. Using a Miltenyi BiotecAutoMACS magnetic cell sorter spleen cells were first labeled with CD4⁺T-cell isolation kit II and the unlabeled fraction was depleted of CD25⁺cells with CD25 depletion kit. The purified B6 cells were stained withan intracellular dye, Carboxyfluorescein Succinimidyl Ester (CFSE),before being admixed at equal concentrations with the purified OTIIcells. They were then injected intravenously (i.v.) intoB6.SJL-Ptprc^(a)/BoyAi (CD45.1) recipient mice.

The next day the recipient CD45.1 mice were treated with targetedtolerogenic synthetic vaccine particles (t²SVP). They were loaded withcombinations of ovalbumin peptide (323-339) (OVA³²³⁻³³⁹), Rapamycin(Rapa) and/or Simvastatin (Simva) and were administered subcutaneously(s.c.).

The injection constitutes a tolerogenic treatment and was followed by 4more injections each spaced 2 weeks apart. After the treatment schedulewas completed the recipient CD45.1 animals were killed and their spleensand popliteal lymph nodes were harvested, mechanically dissociated andfiltered separately through a 70 μM sieve to yield a single-cellsuspension. The spleen cells were depleted of red blood cells (RBCs) byincubation with RBC lysis buffer (Stem Cell Technologies) and cellcounts were performed on both the spleens and lymph nodes.

Spleen or lymph node cells were cultured in CM (complete media)supplemented with 10 U/ml IL-2, restimulated with OPII at 0.3×10⁶cells/well in 96-well round bottom (RB) plates and incubated at 37° C.,5% CO₂. Cells were split at Day 2 and harvested on Day 5. Supernatantswere collected and frozen while cells were stained for phenotypicanalysis by flow cytometry. The cells were analyzed on a BectonDickinson FacsCanto flow cytometer.

In Vivo Administration 2

Spleens from B6.Cg-Tg(TcraTcrb)425Cbn/J (OTII) and C57BL/6 (B6) micewere harvested, mechanically dissociated and filtered separately througha 70 μM sieve to yield a single-cell suspension. Purified CD4⁺CD25−cells were then extracted in a 2-step process using a Miltenyi BiotecAutoMACS magnetic cell sorter. Spleen cells were labeled usingMiltenyi's CD4⁺ T-cell isolation kit II. The unlabeled CD4+ T-cellfraction was then depleted of CD25⁺ cells with CD25 depletion kit. Thepurified CD4 cells from B6 mice were then stained with an intracellulardye, Carboxyfluorescein Succinimidyl Ester (CFSE), before being admixedat equal concentrations with the purified OTII cells. They were theninjected intravenously (i.v.) into B6.SJL-Ptprc^(a)/BoyAi (CD45.1)recipient mice.

The next day the recipient CD45.1 mice were treated with targetedtolerogenic synthetic vaccine particles. They comprised combinations ofovalbumin peptide (323-339) (OVA³²³⁻³³⁹), Rapamycin (Rapa) andSimvastatin (Simva) and were administered subcutaneously (s.c.) orintravenously (i.v.).

After the treatment schedule was completed the recipient CD45.1 animalswere killed and their spleens and popliteal lymph nodes were harvested,mechanically dissociated and filtered separately through a 70 μM sieveto yield a single-cell suspension. The spleen cells were depleted of redblood cells (RBCs) by incorporation with RBC lysis buffer (Stem CellTechnologies) and cell counts were performed on both the spleens andlymph nodes.

Spleen or lymph node cells were cultured in CM supplemented with 10 U/mlIL-2, restimulated with 1 μM OPII at 0.3×10⁶ cells/well in 96-well roundbottom (RB) plates and incubated at 37° C., 5% CO₂. Cells were split atDay 2 and harvested on Day 5. Supernatants were collected and frozenwhile cells were stained for phenotypic analysis by flow cytometry. Thecells were analyzed on a Becton Dickinson FacsCanto flow cytometer.

Results

The results are shown in FIGS. 2 and 3 (Immunomodulator 1: rapamycin;immunomodulator 2: simvastatin). The figures shows in vivo effects anddemonstrates that antigen-specific expansion of effector immune cells isreduced with synthetic nanocarriers comprising antigen andimmunosuppressants as compared to antigen alone or syntheticnanocarriers comprising antigen with and without an immunostimulatorymolecule.

Example 13 Tolerogenic Immune Responses with Synthetic NanocarriersMaterials and Methods Nanocarrier 1

Ovalbumin protein was purchased from Worthington Biochemical Corporation(730 Vassar Avenue, Lakewood, N.J. 08701; Product Code 3048). PLGA witha lactide:glycolide ratio of 3:1 and an inherent viscosity of 0.75 dL/gwas purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,Birmingham, Ala. 35211; Product Code 7525 DLG 7A). Polyvinyl alcohol(85-89% hydrolyzed) was purchased from EMD Chemicals (Product Number1.41350.1001). PLA-PEG block co-polymer with a PEG block ofapproximately 5,000 Da and PLA block of approximately 20,000 Da wassynthesized. Sodium cholate hydrate was purchased from Sigma-AldrichCorp. (3050 Spruce Street, St. Louis, Mo. 63103; Product Code C6445).

Solutions were Prepared as Follows:

Solution 1: Ovalbumin @ 50 mg/mL in phosphate buffered saline solution.The solution was prepared by dissolving ovalbumin in phosphate bufferedsaline solution at room temperature. Solution 2: PLGA @ 100 mg/mL inmethylene chloride. The solution was prepared by dissolving PLGA in puremethylene chloride. Solution 3: PLA-PEG @ 100 mg/mL in methylenechloride. The solution was prepared by dissolving PLA-PEG in puremethylene chloride. Solution 4: Polyvinyl alcohol @ 50 mg/mL and sodiumcholate hydrate @ 10 mg/mL in 100 mM pH 8 phosphate buffer.

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining solution 1 (0.2 mL), solution 2 (0.75 mL), and solution 3(0.25 mL) in a small pressure tube and sonicating at 50% amplitude for40 seconds using a Branson Digital Sonifier 250. A secondary emulsion(W1/O1/W2) was then prepared by combining solution 4 (3.0 mL) with theprimary W1/O1 emulsion, vortexing for 10 s, and sonicating at 30%amplitude for 60 seconds using the Branson Digital Sonifier 250. TheW1/O1/W2 emulsion was added to a beaker containing 70 mM pH 8 phosphatebuffer solution (30 mL) and stirred at room temperature for 2 hours toallow the methylene chloride to evaporate and for the nanocarriers toform. A portion of the nanocarriers were washed by transferring thenanocarrier suspension to a centrifuge tube and centrifuging at 75,600×gand 4° C. for 35 min, removing the supernatant, and re-suspending thepellet in phosphate buffered saline. The washing procedure was repeated,and the pellet was re-suspended in phosphate buffered saline for a finalnanocarrier dispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountof protein in the nanocarrier was determined by an o-phthalaldehydefluorometric assay. The total dry-nanocarrier mass per mL of suspensionwas determined by a gravimetric method.

Effective Diameter Protein Content Nanocarrier ID (nm) (% w/w) 1 19110.1

Nanocarrier 2

Ovalbumin protein was purchased from Worthington Biochemical Corporation(730 Vassar Avenue, Lakewood, N.J. 08701; Product Code 3048). Rapamycinwas purchased from TSZ CHEM (185 Wilson Street, Framingham, Mass. 01702;Product Catalogue # R1017). PLGA with a lactide:glycolide ratio of 3:1and an inherent viscosity of 0.75 dL/g was purchased from SurModicsPharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211; ProductCode 7525 DLG 7A). PLA-PEG block co-polymer with a PEG block ofapproximately 5,000 Da and PLA block of approximately 20,000 Da wassynthesized. Polyvinyl alcohol (85-89% hydrolyzed) was purchased fromEMD Chemicals (Product Number 1.41350.1001). Sodium cholate hydrate waspurchased from Sigma-Aldrich Corp. (3050 Spruce Street, St. Louis, Mo.63103; Product Code C6445).

Solutions were Prepared as Follows:

Solution 1: Ovalbumin @ 50 mg/mL in phosphate buffered saline solution.The solution was prepared by dissolving ovalbumin in phosphate bufferedsaline solution at room temperature. Solution 2: Rapamycin @ 50 mg/mL inmethylene chloride. The solution was prepared by dissolving rapamycin inpure methylene chloride. Solution 3: PLGA @ 100 mg/mL in methylenechloride. The solution was prepared by dissolving PLGA in pure methylenechloride. Solution 4: PLA-PEG @ 100 mg/mL in methylene chloride. Thesolution was prepared by dissolving PLA-PEG in pure methylene chloride.Solution 5: Polyvinyl alcohol @ 50 mg/mL and sodium cholate hydrate @ 10mg/mL in 100 mM pH 8 phosphate buffer.

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining solution 1 (0.2 mL), solution 2 (0.2 mL), solution 3 (0.75mL), and solution 4 (0.25 mL) in a small pressure tube and sonicating at50% amplitude for 40 seconds using a Branson Digital Sonifier 250. Asecondary emulsion (W1/O1/W2) was then prepared by combining solution 5(3.0 mL) with the primary W1/O1 emulsion, vortexing for 10 s, andsonicating at 30% amplitude for 60 seconds using the Branson DigitalSonifier 250. The W1/O1/W2 emulsion was added to a beaker containing 70mM pH 8 phosphate buffer solution (30 mL) and stirred at roomtemperature for 2 hours to allow the methylene chloride to evaporate andfor the nanocarriers to form. A portion of the nanocarriers were washedby transferring the nanocarrier suspension to a centrifuge tube andcentrifuging at 75,600×g and 4° C. for 35 min, removing the supernatant,and re-suspending the pellet in phosphate buffered saline. The washingprocedure was repeated, and the pellet was re-suspended in phosphatebuffered saline for a final nanocarrier dispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountof rapamycin in the nanocarrier was determined by HPLC analysis. Theamount of protein in the nanocarrier was determined by ano-phthalaldehyde fluorometric assay. The total dry-nanocarrier mass permL of suspension was determined by a gravimetric method.

Effective Rapamycin Content Protein Content Nanocarrier ID Diameter (nm)(% w/w) (% w/w) 2 172 7.4 7.6

Immunization

The purpose of this experiment was to assess the effects of encapsulated(t²SVP) immunosuppressant on ongoing antibody responses by measuringantigen-specific immunogloblulins. One group of animals remainedunimmunized as a control. Two groups of animals were immunized using“passive administration” of Ovalbumin or active immunization with OVAand CpG with 3 injections (d0, d14 and d28) followed by an assessment ofantibody titers and one or two weeks of rest. Another two groupsreceived the same immunizations but received boosts every 2 weeks at thesame time they received the treatment. Each of these groups was splitinto three subgroups to test the capacity of different treatments tomodify the Ig titers induced. A control subgroup did not receivetolerogenic treatment. Two other treatments were applied to the othersubgroups including NP carrying just OVA protein or in combination withimmunosuppressant.

For immunization, animals received 20 μl/limb of OVA+CpG (12.5 μg OVA+10μg CpG), both hind limbs s.c. or 25 μg of OVA i.v. in 100 μl. Thetolerogenic treatment included administration of 200 μl t²SVP i.v. using10 μg of OVA. Nanoparticles were provided at 500 μm/ml of OVA content.t²SVP was diluted in such a manner that the same amounts of OVA wereinjected in all groups

Measurement of IgG

The level of IgG antibodies were measured. Blocker Casein in PBS (ThermoFisher, Catalog #37528) was used as diluent. 0.05% Tween-20 in PBS wasused as wash buffer, prepared by adding 10 ml of Tween-20 ((Sigma,Catalog #P9416-100 mL) to 2 liters of a 10×PBS stock (PBS: OmniPur®10×PBS Liquid Concentrate, 4 L, EMD Chemicals, Catalog #6505) and 18Liters of deionized water.

OVA protein at a stock concentration of 5 mg/ml was used as a coatingmaterial. A 1:1000 dilution to 5 μg/ml was used as a workingconcentration. Each well of the assay plates was coated with 100 μldiluted OVA per well, plates were sealed with sealing film (VWR catalog#60941-120), and incubated overnight at 4° C. Costar9017 96-well Flatbottom plates were used as assay plates, Costar9017.

Low-binding polypropylene 96-well plate or tubes were used as set-upplates, in which samples were prepared before being transferred to theassay plate. The setup plates did not contain any antigen and,therefore, serum antibodies did not bind to the plate during the setupof the samples. Setup plates were used for sample preparation tominimize binding that might occur during preparation or pipetting ofsamples if an antigen-coated plate was used to prepare the samples.Before preparing samples in the setup plate, wells were covered withdiluent to block any non-specific binding and the plate was sealed andincubated at 4° C. overnight.

Assay plates were washed three times with wash buffer, and wash bufferwas completely aspirated out of the wells after the last wash. Afterwashing, 300 μl diluent were added to each well of assay plate(s) toblock non-specific binding and plates were incubated at least 2 hours atroom temperature. Serum samples were prepared in the setup plate atappropriate starting dilutions. Starting dilutions were sometimes alsoprepared in 1.5 ml tubes using diluent. Appropriate starting dilutionswere determined based on previous data, where available. Where noprevious data was available, the lowest starting dilution was 1:40. Oncediluted, 200 μl of the starting dilution of the serum sample wastransferred from to the appropriate well of the setup plate.

An exemplary setup plate layout is described as follows: Columns 2 and11 contained anti-Ovabumin monoclonal IgG2b isotype (AbCam, ab17291)standard, diluted to 1 μg/mL (1:4000 dilution). Columns 3-10 containedserum samples (at appropriate dilutions). Columns 1 and 12 were not usedfor samples or standards to avoid any bias of measurements due to edgeeffect. Instead, columns 1 and 12 contained 200 μl diluent. Normal mouseserum diluted 1:40 was used as a negative control. Anti-mouse IgG2adiluted 1:500 from 0.5 mg/mL stock (BD Bioscience) was used as anisotype control.

Once all samples were prepared in the setup plate, the plate was sealedand stored at 4° C. until blocking of the assay plates was complete.Assay plates were washed three times with wash buffer, and wash bufferwas completely aspirated after the last wash. After washing, 100 μL ofdiluent was added to all wells in rows B-H of the assay plates. A12-channel pipet was used to transfer samples from the setup plate tothe assay plate. Samples were mixed prior to transfer by pipetting 150μl of diluted serum up and down 3 times. After mixing, 150 μl of eachsample was transferred from the setup plate and added to row A of therespective assay plate.

Once the starting dilutions of each sample were transferred from thesetup plate to row A of the assay plate, serial dilutions were pipettedon the assay plate as follows: 50 μl of each serum sample was removedfrom row A using 12-channel pipet and mixed with the 100 μl of diluentpreviously added to each well of row B. This step was repeated down theentire plate. After pipetting the dilution of the final row, 50 μl offluid was removed from the wells in the final row and discarded,resulting in a final volume of 100 μl in every well of the assay plate.Once sample dilutions were prepared in the assay plates, the plates wereincubated at room temperature for at least 2 hours.

After the incubation, plates were washed three times with wash buffer.Detection antibody (Goat anti-mouse anti-IgG, HRP conjugated, AbCamab98717) was diluted 1:1500 (0.33 μg/mL) in diluent and 100 μl of thediluted antibody was added to each well. Plates were incubated for 1hour at room temperature and then washed three times with wash buffer,with each washing step including a soak time of at least 30 seconds.

After washing, detection substrate was added to the wells. Equal partsof substrate A and substrate B (BD Biosciences TMB Substrate ReagentSet, catalog #555214) were combined immediately before addition to theassay plates, and 100 μl of the mixed substrate solution were added toeach well and incubated for 10 minutes in the dark. The reaction wasstopped by adding 50 μl of stop solution (2N H2SO4) to each well afterthe 10 minute period. The optical density (OD) of the wells was assessedimmediately after adding the stop solution on a plate reader at 450 nmwith subtraction at 570 nm. Data analysis was performed using MolecularDevice's software SoftMax Pro v5.4. In some cases, a four-parameterlogistic curve-fit graph was prepared with the dilution on the x-axis(log scale) and the OD value on the y-axis (linear scale), and the halfmaximum value (EC50) for each sample was determined. The plate templateat the top of the layout was adjusted to reflect the dilution of eachsample (1 per column).

Results

FIG. 4 shows a descrease in antigen-specific antibody production withnanocarriers comprising peptide antigen and immunosuppressant ascompared to nanocarriers comprising the peptide alone. Panel 3 showsthat the use of a strong immune stimulator, CpG, countered thetolerogenic effects of the synthetic nanocarriers comprising rapamycinin some instances.

Example 14 Tolerogenic Immune Responses with Synthetic NanocarriersMaterials and Methods

Nanocarriers were prepared as in the above example (Example 13).

Immunization

The purpose of this experiment was to assess the effects of encapsulated(t²SVP) immunosuppressant on emerging antibody responses by measuringantigen-specific immunogloblulins in animals that receive the immunogenand NP-treatment at the same time. One group of animals remainedunimmunized as a control (but received the treatment). A second group ofanimals was immunized using “passive administration” of Ovalbumin, and athird group was immunized with OVA and CpG in the sub-scapular region.Each of these groups were given biweekly injection of nanoparticles(NPs) and anti-OVA Ig levels were monitored on the day previous to theboost. For immunization, animals received 100 μl of OVA+CpG s.c.(subscapular) or 25 μg of OVA i.v. in 100 μl. Tolerogenic treatmentcomprised administration of 100 μl t²SVP i.v. Nanocarriers were providedat 5 mg/ml. t2SVP was diluted in such a manner that the same amounts ofOVA were injected in all groups. Injections were performed at d0, d14,d28, d42, d56.

Measurement of IgG

The level of IgG antibodies were measured. Blocker Casein in PBS (ThermoFisher, Catalog #37528) was used as diluent. 0.05% Tween-20 in PBS wasused as wash buffer, prepared by adding 10 ml of Tween-20 ((Sigma,Catalog #P9416-100 mL) to 2 liters of a 10×PBS stock (PBS: OmniPur®10×PBS Liquid Concentrate, 4 L, EMD Chemicals, Catalog #6505) and 18Liters of deionized water.

OVA protein at a stock concentration of 5 mg/ml was used as a coatingmaterial. A 1:1000 dilution to 5 μg/ml was used as a workingconcentration. Each well of the assay plates was coated with 100 μldiluted OVA per well, plates were sealed with sealing film (VWR catalog#60941-120), and incubated overnight at 4° C. Costar9017 96-well Flatbottom plates were used as assay plates, Costar9017.

Low-binding polypropylene 96-well plate or tubes were used as set-upplates, in which samples were prepared before being transferred to theassay plate. The setup plates did not contain any antigen and,therefore, serum antibodies did not bind to the plate during the setupof the samples. Setup plates were used for sample preparation tominimize binding that might occur during preparation or pipetting ofsamples if an antigen-coated plate was used to prepare the samples.Before preparing samples in the setup plate, wells were covered withdiluent to block any non-specific binding and the plate was sealed andincubated at 4° C. overnight.

Assay plates were washed three times with wash buffer, and wash bufferwas completely aspirated out of the wells after the last wash. Afterwashing, 300 μl diluent were added to each well of assay plate(s) toblock non-specific binding and plates were incubated at least 2 hours atroom temperature. Serum samples were prepared in the setup plate atappropriate starting dilutions. Starting dilutions were sometimes alsoprepared in 1.5 ml tubes using diluent. Appropriate starting dilutionswere determined based on previous data, where available. Where noprevious data was available, the lowest starting dilution was 1:40. Oncediluted, 200 μl of the starting dilution of the serum sample wastransferred from to the appropriate well of the setup plate.

An exemplary setup plate layout is described as follows: Columns 2 and11 contained anti-Ovabumin monoclonal IgG2b isotype (AbCam, ab17291)standard, diluted to 1 μg/mL (1:4000 dilution). Columns 3-10 containedserum samples (at appropriate dilutions). Columns 1 and 12 were not usedfor samples or standards to avoid any bias of measurements due to edgeeffect. Instead, columns 1 and 12 contained 200 μl diluent. Normal mouseserum diluted 1:40 was used as a negative control. Anti-mouse IgG2adiluted 1:500 from 0.5 mg/mL stock (BD Bioscience) was used as anisotype control.

Once all samples were prepared in the setup plate, the plate was sealedand stored at 4° C. until blocking of the assay plates was complete.Assay plates were washed three times with wash buffer, and wash bufferwas completely aspirated after the last wash. After washing, 100 μL ofdiluent was added to all wells in rows B-H of the assay plates. A12-channel pipet was used to transfer samples from the setup plate tothe assay plate. Samples were mixed prior to transfer by pipetting 150μl of diluted serum up and down 3 times. After mixing, 150 μl of eachsample was transferred from the setup plate and added to row A of therespective assay plate.

Once the starting dilutions of each sample were transferred from thesetup plate to row A of the assay plate, serial dilutions were pipettedon the assay plate as follows: 50 μl of each serum sample was removedfrom row A using 12-channel pipet and mixed with the 100 μl of diluentpreviously added to each well of row B. This step was repeated down theentire plate. After pipetting the dilution of the final row, 50 μl offluid was removed from the wells in the final row and discarded,resulting in a final volume of 100 μl in every well of the assay plate.Once sample dilutions were prepared in the assay plates, the plates wereincubated at room temperature for at least 2 hours.

After the incubation, plates were washed three times with wash buffer.Detection antibody (Goat anti-mouse anti-IgG, HRP conjugated, AbCamab98717) was diluted 1:1500 (0.33 μg/mL) in diluent and 100 μl of thediluted antibody was added to each well. Plates were incubated for 1hour at room temperature and then washed three times with wash buffer,with each washing step including a soak time of at least 30 seconds.

After washing, detection substrate was added to the wells. Equal partsof substrate A and substrate B (BD Biosciences TMB Substrate ReagentSet, catalog #555214) were combined immediately before addition to theassay plates, and 100 μl of the mixed substrate solution were added toeach well and incubated for 10 minutes in the dark. The reaction wasstopped by adding 50 μl of stop solution (2N H2504) to each well afterthe 10 minute period. The optical density (OD) of the wells was assessedimmediately after adding the stop solution on a plate reader at 450 nmwith subtraction at 570 nm. Data analysis was performed using MolecularDevice's software SoftMax Pro v5.4. In some cases, a four-parameterlogistic curve-fit graph was prepared with the dilution on the x-axis(log scale) and the OD value on the y-axis (linear scale), and the halfmaximum value (EC50) for each sample was determined. The plate templateat the top of the layout was adjusted to reflect the dilution of eachsample (1 per column).

Results

FIG. 5 shows a descrease in antigen-specific antibody production withnanocarriers comprising antigen and immunosuppressant as compared tonanocarriers comprising the antigen alone. Again the data also show thatthe use of a strong immune stimulator, CpG, countered the tolerogeniceffects of the synthetic nanocarriers comprising rapamycin in someinstances.

Example 15 Assessing the Effects of Nanocarriers with Antigens andImmunosuppressants on Immune Responses Materials and Methods Nanocarrier1

Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T and Bcell epitope of Ovalbumin protein, was purchased from Bachem AmericasInc. (3132 Kashiwa Street, Torrance Calif. 90505; Part #4065609). PLGAwith a lactide:glycolide ratio of 3:1 and an inherent viscosity of 0.75dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,Birmingham, Ala. 35211; Product Code 7525 DLG 7A). PLA-PEG blockco-polymer with a PEG block of approximately 5,000 Da and PLA block ofapproximately 20,000 Da was synthesized. Polyvinyl alcohol (85-89%hydrolyzed) was purchased from EMD Chemicals (Product Number1.41350.1001).

Solutions were Prepared as Follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13 M hydrochloric acid solution at room temperature.Solution 2: PLGA @ 100 mg/mL in methylene chloride. The solution wasprepared by dissolving PLGA in pure methylene chloride. Solution 3:PLA-PEG @ 100 mg/mL in methylene chloride. The solution was prepared bydissolving PLA-PEG in pure methylene chloride. Solution 4: Polyvinylalcohol @ 50 mg/mL in 100 mM pH 8 phosphate buffer.

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining solution 1 (0.2 mL), solution 2 (0.75 mL), and solution 3(0.25 mL) in a small pressure tube and sonicating at 50% amplitude for40 seconds using a Branson Digital Sonifier 250. A secondary emulsion(W1/O1/W2) was then prepared by combining solution 4 (3.0 mL) with theprimary W1/O1 emulsion, vortexing for 10 s, and sonicating at 30%amplitude for 60 seconds using the Branson Digital Sonifier 250. TheW1/O1/W2 emulsion was added to a beaker containing 70 mM pH 8 phosphatebuffer solution (30 mL) and stirred at room temperature for 2 hours toallow the methylene chloride to evaporate and for the nanocarriers toform. A portion of the nanocarriers were washed by transferring thenanocarrier suspension to a centrifuge tube and centrifuging at 75,600×gand 4° C. for 35 min, removing the supernatant, and re-suspending thepellet in phosphate buffered saline. The washing procedure was repeated,and the pellet was re-suspended in phosphate buffered saline for a finalnanocarrier dispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountof peptide in the nanocarrier was determined by HPLC analysis. The totaldry-nanocarrier mass per mL of suspension was determined by agravimetric method.

Effective Diameter Peptide Content Nanocarrier ID (nm) (% w/w) 1 234 2.1

Nanocarrier 2

Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T and Bcell epitope of Ovalbumin protein, was purchased from Bachem AmericasInc. (3132 Kashiwa Street, Torrance Calif. 90505; Part #4065609).Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham,Mass. 01702; Product Catalogue # R1017). PLGA with a lactide:glycolideratio of 3:1 and an inherent viscosity of 0.75 dL/g was purchased fromSurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211;Product Code 7525 DLG 7A). PLA-PEG block co-polymer with a PEG block ofapproximately 5,000 Da and PLA block of approximately 20,000 Da wassynthesized. Polyvinyl alcohol (85-89% hydrolyzed) was purchased fromEMD Chemicals (Product Number 1.41350.1001).

Solutions were Prepared as Follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13 M hydrochloric acid solution at room temperature.Solution 2: Rapamycin @ 50 mg/mL in methylene chloride. The solution wasprepared by dissolving rapamycin in pure methylene chloride. Solution 3:PLGA @ 100 mg/mL in methylene chloride. The solution was prepared bydissolving PLGA in pure methylene chloride. Solution 4: PLA-PEG @ 100mg/mL in methylene chloride. The solution was prepared by dissolvingPLA-PEG in pure methylene chloride. Solution 5: Polyvinyl alcohol @ 50mg/mL in 100 mM pH 8 phosphate buffer.

A primary water-in-oil emulsion was prepared first. W1/O1 was preparedby combining solution 1 (0.2 mL), solution 2 (0.2 mL), solution 3 (0.75mL), and solution 4 (0.25 mL) in a small pressure tube and sonicating at50% amplitude for 40 seconds using a Branson Digital Sonifier 250. Asecondary emulsion (W1/O1/W2) was then prepared by combining solution 5(3.0 mL) with the primary W1/O1 emulsion, vortexing for 10 s, andsonicating at 30% amplitude for 60 seconds using the Branson DigitalSonifier 250. The W1/O1/W2 emulsion was added to a beaker containing 70mM pH 8 phosphate buffer solution (30 mL) and stirred at roomtemperature for 2 hours to allow the methylene chloride to evaporate andfor the nanocarriers to form. A portion of the nanocarriers were washedby transferring the nanocarrier suspension to a centrifuge tube andcentrifuging at 21,000×g and 4° C. for 45 min, removing the supernatant,and re-suspending the pellet in phosphate buffered saline. The washingprocedure was repeated, and the pellet was re-suspended in phosphatebuffered saline for a final nanocarrier dispersion of about 10 mg/mL.

Nanocarrier size was determined by dynamic light scattering. The amountsof peptide and rapamycin in the nanocarrier were determined by HPLCanalysis. The total dry-nanocarrier mass per mL of suspension wasdetermined by a gravimetric method.

Effective Rapamycin Content Peptide Content Nanocarrier ID Diameter (nm)(% w/w) (% w/w) 2 227 9.0 2.5

Immunization

Animals received immunization every 2 weeks at the same time theyreceived the treatment. Each of these groups was split into subgroups totest the capacity of different treatments to modify the Ig titersinduced. A control subgroup did not receive tolerogenic treatment. Twosubgroups received nanocarrier carrying just OVA₃₂₃₋₃₃₉ peptide or incombination with rapamycin.

Immunization was administered via the following routes (values are peranimal): 20 μl/limb of OVA+CpG (12.5 μg OVA+10 μg CpG), both hind limbss.c. Tolerogenic treatments were administered via the following route(values are per animal): 200 μl nanocarriers were provided at 100 μg/mlof OVA₃₂₃₋₃₃₉ content.

Measurement of IgG

The level of IgG antibodies were measured. Blocker Casein in PBS (ThermoFisher, Catalog #37528) was used as diluent. 0.05% Tween-20 in PBS wasused as wash buffer, prepared by adding 10 ml of Tween-20 ((Sigma,Catalog #P9416-100 mL) to 2 liters of a 10×PBS stock (PBS: OmniPur®10×PBS Liquid Concentrate, 4 L, EMD Chemicals, Catalog #6505) and 18Liters of deionized water.

OVA protein at a stock concentration of 5 mg/ml was used as a coatingmaterial. A 1:1000 dilution to 5 μg/ml was used as a workingconcentration. Each well of the assay plates was coated with 100 μldiluted OVA per well, plates were sealed with sealing film (VWR catalog#60941-120), and incubated overnight at 4° C. Costar9017 96-well Flatbottom plates were used as assay plates, Costar9017.

Low-binding polypropylene 96-well plate or tubes were used as set-upplates, in which samples were prepared before being transferred to theassay plate. The setup plates did not contain any antigen and,therefore, serum antibodies did not bind to the plate during the setupof the samples. Setup plates were used for sample preparation tominimize binding that might occur during preparation or pipetting ofsamples if an antigen-coated plate was used to prepare the samples.Before preparing samples in the setup plate, wells were covered withdiluent to block any non-specific binding and the plate was sealed andincubated at 4° C. overnight.

Assay plates were washed three times with wash buffer, and wash bufferwas completely aspirated out of the wells after the last wash. Afterwashing, 300 μl diluent were added to each well of assay plate(s) toblock non-specific binding and plates were incubated at least 2 hours atroom temperature. Serum samples were prepared in the setup plate atappropriate starting dilutions. Starting dilutions were sometimes alsoprepared in 1.5 ml tubes using diluent. Appropriate starting dilutionswere determined based on previous data, where available. Where noprevious data was available, the lowest starting dilution was 1:40. Oncediluted, 200 μl of the starting dilution of the serum sample wastransferred from to the appropriate well of the setup plate.

An exemplary setup plate layout is described as follows: Columns 2 and11 contained anti-Ovabumin monoclonal IgG2b isotype (AbCam, ab17291)standard, diluted to 1 μg/mL (1:4000 dilution). Columns 3-10 containedserum samples (at appropriate dilutions). Columns 1 and 12 were not usedfor samples or standards to avoid any bias of measurements due to edgeeffect. Instead, columns 1 and 12 contained 200 μl diluent. Normal mouseserum diluted 1:40 was used as a negative control. Anti-mouse IgG2adiluted 1:500 from 0.5 mg/mL stock (BD Bioscience) was used as anisotype control.

Once all samples were prepared in the setup plate, the plate was sealedand stored at 4° C. until blocking of the assay plates was complete.Assay plates were washed three times with wash buffer, and wash bufferwas completely aspirated after the last wash. After washing, 100 μL ofdiluent was added to all wells in rows B-H of the assay plates. A12-channel pipet was used to transfer samples from the setup plate tothe assay plate. Samples were mixed prior to transfer by pipetting 150μl of diluted serum up and down 3 times. After mixing, 1500 of eachsample was transferred from the setup plate and added to row A of therespective assay plate.

Once the starting dilutions of each sample were transferred from thesetup plate to row A of the assay plate, serial dilutions were pipettedon the assay plate as follows: 50 μl of each serum sample was removedfrom row A using 12-channel pipet and mixed with the 100 μl of diluentpreviously added to each well of row B. This step was repeated down theentire plate. After pipetting the dilution of the final row, 50 μl offluid was removed from the wells in the final row and discarded,resulting in a final volume of 100 μl in every well of the assay plate.Once sample dilutions were prepared in the assay plates, the plates wereincubated at room temperature for at least 2 hours.

After the incubation, plates were washed three times with wash buffer.Detection antibody (Goat anti-mouse anti-IgG, HRP conjugated, AbCamab98717) was diluted 1:1500 (0.33 μg/mL) in diluent and 100 μl of thediluted antibody was added to each well. Plates were incubated for 1hour at room temperature and then washed three times with wash buffer,with each washing step including a soak time of at least 30 seconds.

After washing, detection substrate was added to the wells. Equal partsof substrate A and substrate B (BD Biosciences TMB Substrate ReagentSet, catalog #555214) were combined immediately before addition to theassay plates, and 100 μl of the mixed substrate solution were added toeach well and incubated for 10 minutes in the dark. The reaction wasstopped by adding 50 μl of stop solution (2N H2SO4) to each well afterthe 10 minute period. The optical density (OD) of the wells was assessedimmediately after adding the stop solution on a plate reader at 450 nmwith subtraction at 570 nm. Data analysis was performed using MolecularDevice's software SoftMax Pro v5.4. In some cases, a four-parameterlogistic curve-fit graph was prepared with the dilution on the x-axis(log scale) and the OD value on the y-axis (linear scale), and the halfmaximum value (EC50) for each sample was determined. The plate templateat the top of the layout was adjusted to reflect the dilution of eachsample (1 per column).

Determination of % OVA+Dividing B Cells

Ovalbumin+ B-cell division was assessed by flow cytometry. Splenocytesfrom experimental animals were stained with Cell Tracker Orange (CTO), athiol-reactive fluorescent probe suitable for long-term cell labeling,and cultured in complete media at 37 C, 5% CO₂ with Ovalbumin protein orpeptide for 3 days. On day 3 the cells were washed, blocked withanti-CD16/32 antibody and then stained with conjugated antibodiesspecific to B220 and CD19. Alexa 647 conjugated ovalbumin protein wasalso incubated with the cells to label Ovalbumin specific BCRs. Thosesplenocytes that were CD19+B220+OVA-Alexa647+ were assessed forproliferation by comparing the differential CTO staining. Those thatwere CTO low were labeled as proliferating Ovalbumin+B-cells and werecompared to the CTO high Ovalbumin+B-cells to quantify the percentages.

Results

FIG. 6 shows a reduction in antigen-specific IgG levels with theadministration of synthetic nanocarriers comprising ova peptide and theimmunosuppressant rapamycin. FIG. 7 also demonstrates a reduction, butin the number of antigen-specific B cells with the syntheticnanocarriers. These results demonstrate the reduction in undesiredimmune responses with synthetic nanocarriers coupled to ova peptide(comprising an MHC Class II-restricted epitope) and immunosuppressant.There results are relevant in any context where the reduction ofantigen-specific B cells would provide a benefit.

Example 16 Assessing the Effects of Nanocarriers with Antigens andImmunosuppressants Nanocarriers

Nanocarriers were prepared according to methods provided above (Example15).

Immunization

The nanocarriers were thawed and equilibrated. Initial dilutionsconstituted a 10× stock solution, and were further diluted to aconcentration of 100 μg/ml in OVA₃₂₃₋₃₃₉, or a 1× solution. This 1×solution was used for injections at 200 μl per i.v. injection. Animalswere immunized with OVA protein (OVA) and treated with OVA₃₂₃₋₃₃₉peptide to assess the capacity of nanocarriers to control the immuneresponses in absence of B cell antigens. Immunization routes were asfollows: 10 μg of OVA+4 mg Alum i.p. in 400 μl per each Balb/Cimmunologically naïve female mouse. Experimental groups consisted of 5animals each. Spleen cells were restimulated with antigen using CFSE orCTO to determine the amount of Ag-specific proliferation.

Levels of Specific Types of Immune Cells

FCS files were analyzed using FlowJo software. 7AAD positive cells (anuclear dye that label dead cells) positive cells were excluded and cellmorphologies dependent on expression of CD4, CD8, Gr-1, F4/80, B220,TCRb and CD11b were quantified.

Gating strategy for T-cell subsets→7AAD−F4/80−GR-1−TCRb+CD4+/−CD8+/−Gating strategy for B-cell subsets→7AAD−B220+TCRb−Gating strategy for Eosinophils→7AAD−F4/80−Gr-1+TCRb−CD11b+Gr-1+

Determination of % Dividing CD4+ T Cells

The frequency of Ovalbumin reactive CD4⁺ T cells was calculated by wayof flow cytometry. Splenocytes from experimental animals were stainedwith CFSE, a thiol-reactive Fluorescent Probe suitable for long-termcell labeling, and cultured in complete media at 37 C, 5% CO₂ withOvalbumin protein for 3 days. On day 3 the cells were washed, blockedwith anti-CD16/32 antibody and then stained with conjugated antibodiesspecific to TCR CD4 and CD8a. Splenocytes that were TCR+CD4 or TCR+CD8a+were assessed for proliferation by comparing the differential CFSEstaining.

Results

FIGS. 8 and 9 demonstrate the effectiveness of the nanocarriers in ananimal model. Specifically, FIG. 8 demonstrates a reduction in thenumber of CD4+ T cells in lavage samples from animal subjects treatedwith synthetic nanocarriers comprising OVA₃₂₃₋₃₃₉ (an MHC ClassII-restricted epitope) and immunosuppressant. FIG. 9 demonstrates areduction in the percentage of dividing CD4+ T cells as a result of thesame treatment.

What is claimed is:
 1. A composition comprising: (i) a first populationof synthetic nanocarriers that are coupled to immunosuppressants, and(ii) a second population of synthetic nanocarriers that are coupled totherapeutic protein APC presentable antigens.
 2. The composition ofclaim 1, wherein the first population and second population are thesame.
 3. The composition of claim 1 or 2, wherein the immunosuppressantscomprise a statin, an mTOR inhibitor, a TGF-β signaling agent, acorticosteroid, an inhibitor of mitochondrial function, a P38 inhibitor,an NF-κβ inhibitor, an adenosine receptor agonist, a prostaglandin E2agonist, a phosphodiesterasse 4 inhibitor, an HDAC inhibitor or aproteasome inhibitor.
 4. The composition of claim 4, wherein the mTORinhibitor is rapamycin.
 5. The composition of any of claims 1-4, whereinthe therapeutic protein APC presentable antigens comprise MHC ClassI-restricted and/or MHC Class II-restricted epitopes of a therapeuticprotein.
 6. The composition of claim 5, wherein the therapeutic proteinAPC presentable antigens comprise MHC Class II-restricted epitopes of atherapeutic protein.
 7. The composition of any of claims 1-6, whereinthe therapeutic protein APC presentable antigens comprise B cellepitopes of a therapeutic protein.
 8. The composition of any of claims1-6, wherein the therapeutic protein APC presentable antigens comprisesubstantially no B cell epitopes of a therapeutic protein.
 9. Thecomposition of any of claims 1-8, wherein the therapeutic protein APCpresentable antigens comprise a therapeutic protein for proteinreplacement or protein supplementation therapy, or a fragment thereof.10. The composition of any of claims 1-9, wherein the therapeuticprotein APC presentable antigens comprise a/an infusible or injectabletherapeutic protein, enzyme, enzyme cofactor, hormone, blood or bloodcoagulation factor, cytokine, interferon, growth factor, monoclonalantibody, polyclonal antibody or protein associated with Pompe'sdisease, or a fragment thereof.
 11. The composition of claim 10, whereinthe infusible or injectable therapeutic protein comprises Tocilizumab,alpha-1 antitrypsin, Hematide, albinterferon alfa-2b, Rhucin,tesamorelin, ocrelizumab, belimumab, pegloticase, taliglucerase alfa,agalsidase alfa or velaglucerase alfa.
 12. The composition of claim 10,where the enzyme comprises an oxidoreductase, transferase, hydrolase,lyase, isomerase or ligase.
 13. The composition of claim 10, whereinenzyme comprises an enzyme for enzyme replacement therapy for alysosomal storage disorder.
 14. The composition of claim 13, wherein theenzyme for enzyme replacement therapy for a lysosomal storage disordercomprises imiglucerase, a-galactosidase A (a-gal A), agalsidase beta,acid a-glucosidase (GAA), alglucosidase alfa, LUMIZYME, MYOZYME,arylsulfatase B, laronidase, ALDURAZYME, idursulfase, ELAPRASE,arylsulfatase B or NAGLAZYME.
 15. The composition of claim 10, whereinthe cytokine comprises a lymphokine, interleukin, chemokine, type 1cytokine or a type 2 cytokine.
 16. The composition of claim 10, whereinthe blood and blood coagulation factor comprises Factor I, Factor II,tissue factor, Factor V, Factor VII, Factor VIII, Factor IX, Factor X,Factor Xa, Factor XII, Factor XIII, von Willebrand factor,prekallikrein, high-molecular weight kininogen, fibronectin,antithrombin III, heparin cofactor II, protein C, protein S, protein Z,protein Z-related protease inhibitor (ZPI), plasminogen, alpha2-antiplasmin, tissue plasminogen activator (tPA), urokinase,plasminogen activator inhibitor-1 (PAI1), plasminogen activatorinhibitor-2 (PAI2), cancer procoagulant or epoetin alfa.
 17. Thecomposition of any of claims 1-16, wherein the composition is in anamount effective to generate a tolerogenic immune response to thetherapeutic protein APC presentable antigen.
 18. The composition of anyof claims 1-17, wherein the composition is in an amount effective toreduce the generation of therapeutic protein-specific antibodies and/orCD4+ T cell proliferation and/or activity and/or B cell proliferationand/or activity when administered to a subject.
 19. The composition ofany of claims 1-18, wherein the load of the immunosuppressant and/ortherapeutic protein APC presentable antigen on average across the firstand/or second population of synthetic nanocarriers is between 0.0001%and 50%.
 20. The composition of claim 19, wherein the load of theimmunosuppressant and/or therapeutic protein APC presentable antigen onaverage across the first and/or second population of syntheticnanocarriers is between 0.1% and 10%.
 21. The composition of any ofclaims 1-20, wherein the synthetic nanocarriers of the first populationand/or second population comprise lipid nanoparticles, polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, buckyballs, nanowires, virus-like particles or peptide orprotein particles.
 22. The composition of claim 21, wherein thesynthetic nanocarriers of the first and/or second populations compriselipid nanoparticles.
 23. The composition of claim 22, wherein thesynthetic nanocarriers of the first and/or second populations compriseliposomes.
 24. The composition of claim 21, wherein the syntheticnanocarriers of the first and/or second populations comprise metallicnanoparticles.
 25. The composition of claim 24, wherein the metallicnanoparticles comprise gold nanoparticles.
 26. The composition of claim21, wherein the synthetic nanocarriers of the first and/or secondpopulations comprise polymeric nanoparticles.
 27. The composition ofclaim 26, wherein the polymeric nanoparticle comprises polymer that is anon-methoxy-terminated, pluronic polymer.
 28. The composition of claim26 or 27, wherein the polymeric nanoparticles comprise a polyester, apolyester coupled to a polyether, polyamino acid, polycarbonate,polyacetal, polyketal, polysaccharide, polyethyloxazoline orpolyethyleneimine.
 29. The composition of claim 28, wherein thepolyester comprises a poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid) or polycaprolactone.
 30. The compositionof claim 28 or 29, wherein the polymeric nanoparticles comprise apolyester and a polyester coupled to a polyether.
 31. The composition ofany of claims 28-30, wherein the polyether comprises polyethylene glycolor polypropylene glycol.
 32. The composition of any of claims 1-31,wherein the mean of a particle size distribution obtained using dynamiclight scattering of the synthetic nanocarriers of the first and/orsecond population is a diameter greater than 100 nm.
 33. The compositionof claim 32, wherein the diameter is greater than 150 nm.
 34. Thecomposition of claim 33, wherein the diameter is greater than 200 nm.35. The composition of claim 34, wherein the diameter is greater than250 nm.
 36. The composition of claim 35, wherein the diameter is greaterthan 300 nm.
 37. The composition of any of claims 1-36, wherein theaspect ratio of the synthetic nanocarriers of the first populationand/or second population is greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3,1:5, 1:7 or 1:10.
 38. The composition of any of claims 1-37, wherein thecomposition further comprises a pharmaceutically acceptable excipient.39. A dosage form comprising the composition of any of claims 1-38. 40.A method comprising administering the composition of any of claims 1-38or the dosage form of claim 39 to a subject that is being administeredor will be administered a therapeutic protein.
 41. The method of claim40, wherein the method further comprises administering the therapeuticprotein to the subject.
 42. The method of claim 40 or 41, wherein thetherapeutic protein is administered prior to, concomitantly with orafter the administration of the composition or dosage form.
 43. Themethod of any of claims 40-42, wherein one or more maintenance doses ofthe composition or dosage form is administered to the subject.
 44. Themethod of any of claims 40-43, wherein the method further comprisesassessing the generation of an undesired immune response in the subjectprior to and/or after the administration of the composition or dosageform and/or the therapeutic protein.
 45. The method of claim 44, whereinthe undesired immune response is the generation of therapeuticprotein-specific antibodies and/or CD4+ T cell proliferation and/oractivity and/or B cell proliferation and/or activity.
 46. The method ofany of claims 40-45, wherein the therapeutic protein comprises atherapeutic protein for protein replacement or protein supplementationtherapy.
 47. The method of any of claims 40-45, wherein the therapeuticprotein comprises a/an infusible or injectable therapeutic protein,enzyme, enzyme cofactor, hormone, blood or blood coagulation factor,cytokine, interferon, growth factor, monoclonal antibody, polyclonalantibody or protein associated with Pompe's disease.
 48. The method ofclaim 47, wherein the infusible or injectable therapeutic proteincomprises Tocilizumab, alpha-1 antitrypsin, Hematide, albinterferonalfa-2b, Rhucin, tesamorelin, ocrelizumab, belimumab, pegloticase,taliglucerase alfa, agalsidase alfa or velaglucerase alfa.
 49. Themethod of claim 47, where the enzyme comprises an oxidoreductase,transferase, hydrolase, lyase, isomerase or ligase.
 50. The method ofclaim 47, wherein enzyme comprises an enzyme for enzyme replacementtherapy for a lysosomal storage disorder.
 51. The method of claim 50,wherein the enzyme for enzyme replacement therapy for a lysosomalstorage disorder comprises imiglucerase, a-galactosidase A (a-gal A),agalsidase beta, acid a-glucosidase (GAA), alglucosidase alfa, LUMIZYME,MYOZYME, arylsulfatase B, laronidase, ALDURAZYME, idursulfase, ELAPRASE,arylsulfatase B or NAGLAZYME.
 52. The method of claim 47, wherein thecytokine comprises a lymphokine, interleukin, chemokine, type 1 cytokineor a type 2 cytokine.
 53. The method of claim 47, wherein the blood andblood coagulation factor comprises Factor I, Factor II, tissue factor,Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor Xa,Factor XII, Factor XIII, von Willebrand factor, prekallikrein,high-molecular weight kininogen, fibronectin, antithrombin III, heparincofactor II, protein C, protein S, protein Z, protein Z-related proteaseinhibitor (ZPI), plasminogen, alpha 2-antiplasmin, tissue plasminogenactivator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI1),plasminogen activator inhibitor-2 (PAI2), cancer procoagulant or epoetinalfa.
 54. The method of any of claims 40-53, wherein the administeringof the first and/or second population of synthetic nanocarriers and/ortherapeutic protein is by intravenous, intraperitoneal, transmucosal,oral, subcutaneous, pulmonary, intranasal, intradermal or intramuscularadministration.
 55. The method of claim 54, wherein the administering ofthe first and/or second population of synthetic nanocarriers and/ortherapeutic protein is by inhalation or intravenous, subcutaneous ortransmucosal administration.
 56. A method comprising: administering to asubject a composition comprising: (i) a first population of syntheticnanocarriers that are coupled to immunosuppressants, and (ii) a secondpopulation of synthetic nanocarriers that are coupled to therapeuticprotein APC presentable antigens, wherein the composition is in anamount effective to reduce the generation of an undesired immuneresponse against the therapeutic protein APC presentable antigens.
 57. Amethod comprising: reducing the generation of an undesired immuneresponse in a subject by administering a composition comprising: (i) afirst population of synthetic nanocarriers that are coupled toimmunosuppressants, and (ii) a second population of syntheticnanocarriers that are coupled to therapeutic protein APC presentableantigens.
 58. A method comprising: administering a composition to asubject according to a protocol that was previously shown to reduce thegeneration of an undesired immune response to therapeutic protein APCpresentable antigens in one or more test subjects; wherein thecomposition comprises: (i) a first population of synthetic nanocarriersthat are coupled to immunosuppressants, and (ii) a second population ofsynthetic nanocarriers that are coupled to the therapeutic protein APCpresentable antigens.
 59. The method of any of claims 56-58, wherein thefirst population and second population are the same.
 60. The method ofany of claims 56-59, wherein the method further comprises providing oridentifying the subject.
 61. The method of any of claims 56-60, whereinthe method further comprises assessing the generation of the undesiredimmune response in the subject prior to and/or after the administrationof the composition.
 62. The method of claim 61, wherein the undesiredimmune response is the generation of therapeutic protein-specificantibodies and/or CD4+ T cell proliferation and/or activity and/or Bcell proliferation and/or activity.
 63. The method of any of claims56-62, wherein the immunosuppressants comprise a statin, an mTORinhibitor, a TGF-β signaling agent, a corticosteroid, an inhibitor ofmitochondrial function, a P38 inhibitor, an NF-κβ inhibitor, anadenosine receptor agonist, a prostaglandin E2 agonist, aphosphodiesterasse 4 inhibitor, an HDAC inhibitor or a proteasomeinhibitor.
 64. The method of claim 63, wherein the mTOR inhibitor israpamycin.
 65. The method of any of claims 56-64, wherein thetherapeutic protein APC presentable antigens comprise MHC ClassI-restricted and/or MHC Class II-restricted epitopes of a therapeuticprotein.
 66. The method of claim 65, wherein the therapeutic protein APCpresentable antigens comprise MHC Class II-restricted epitopes of atherapeutic protein.
 67. The method of any of claims 56-66, wherein thetherapeutic protein APC presentable antigens comprise B cell epitopes ofa therapeutic protein.
 68. The method of any of claims 56-67, whereinthe therapeutic protein comprises a therapeutic protein for proteinreplacement or protein supplementation therapy.
 69. The method of any ofclaims 56-68, wherein the therapeutic protein comprises a/an infusibleor injectable therapeutic protein, enzyme, enzyme cofactor, hormone,blood or blood coagulation factor, cytokine, interferon, growth factor,monoclonal antibody, polyclonal antibody or protein associated withPompe's disease.
 70. The method of claim 69, wherein the infusible orinjectable therapeutic protein comprises Tocilizumab, alpha-1antitrypsin, Hematide, albinterferon alfa-2b, Rhucin, tesamorelin,ocrelizumab, belimumab, pegloticase, taliglucerase alfa, agalsidase alfaor velaglucerase alfa.
 71. The method of claim 69, where the enzymecomprises an oxidoreductase, transferase, hydrolase, lyase, isomerase orligase.
 72. The method of claim 69, wherein enzyme comprises an enzymefor enzyme replacement therapy for a lysosomal storage disorder.
 73. Themethod of claim 72, wherein the enzyme for enzyme replacement therapyfor a lysosomal storage disorder comprises imiglucerase, a-galactosidaseA (a-gal A), agalsidase beta, acid a-glucosidase (GAA), alglucosidasealfa, LUMIZYME, MYOZYME, arylsulfatase B, laronidase, ALDURAZYME,idursulfase, ELAPRASE, arylsulfatase B or NAGLAZYME.
 74. The method ofclaim 69, wherein the cytokine comprises a lymphokine, interleukin,chemokine, type 1 cytokine or a type 2 cytokine.
 75. The method of claim69, wherein the blood and blood coagulation factor comprises Factor I,Factor II, tissue factor, Factor V, Factor VII, Factor VIII, Factor IX,Factor X, Factor Xa, Factor XII, Factor XIII, von Willebrand factor,prekallikrein, high-molecular weight kininogen, fibronectin,antithrombin III, heparin cofactor II, protein C, protein S, protein Z,protein Z-related protease inhibitor (ZPI), plasminogen, alpha2-antiplasmin, tissue plasminogen activator (tPA), urokinase,plasminogen activator inhibitor-1 (PAI1), plasminogen activatorinhibitor-2 (PAI2), cancer procoagulant or epoetin alfa.
 76. The methodof any of claims 56-75, wherein the composition is in an amounteffective to reduce the generation of therapeutic protein-specificantibodies and/or CD4+ T cell proliferation and/or activity and/or Bcell proliferation and/or activity.
 77. The method of any of claims56-76, wherein the load of the immunosuppressant and/or therapeuticprotein APC presentable antigen on average across the first populationand/or second population of synthetic nanocarriers is between 0.0001%and 50%.
 78. The method of any of claims 56-76, wherein the load of theimmunosuppressant and/or therapeutic protein APC presentable antigen onaverage across the first population and/or second population ofsynthetic nanocarriers is between 0.1% and 10%.
 79. The method of any ofclaims 56-78, wherein the synthetic nanocarriers of the first populationand/or second population comprise lipid nanoparticles, polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, buckyballs, nanowires, virus-like particles or peptide orprotein particles.
 80. The method of claim 79, wherein the syntheticnanocarriers of the first and/or second populations comprise lipidnanoparticles.
 81. The method of claim 80, wherein the syntheticnanocarriers of the first and/or second populations comprise liposomes.82. The method of claim 79, wherein the synthetic nanocarriers of thefirst and/or second populations comprise metallic nanoparticles.
 83. Themethod of claim 82, wherein the metallic nanoparticles comprise goldnanoparticles.
 84. The method of claim 79, wherein the syntheticnanocarriers of the first and/or second populations comprise polymericnanoparticles.
 85. The method of claim 84, wherein the polymericnanoparticle comprises polymer that is a non-methoxy-terminated,pluronic polymer.
 86. The method of claim 84 or 85, wherein thepolymeric nanoparticles comprise a polyester, a polyester coupled to apolyether, polyamino acid, polycarbonate, polyacetal, polyketal,polysaccharide, polyethyloxazoline or polyethyleneimine.
 87. The methodof claim 86, wherein the polyester comprises a poly(lactic acid),poly(glycolic acid), poly(lactic-co-glycolic acid) or polycaprolactone.88. The method of claim 86 or 87, wherein the polymeric nanoparticlescomprise a polyester and a polyester coupled to a polyether.
 89. Themethod of any of claims 86-88, wherein the polyether comprisespolyethylene glycol or polypropylene glycol.
 90. The method of any ofclaims 56-89, wherein the mean of a particle size distribution obtainedusing dynamic light scattering of the synthetic nanocarriers of thefirst and/or second population is a diameter greater than 100 nm. 91.The method of claim 90, wherein the diameter is greater than 150 nm. 92.The method of claim 91, wherein the diameter is greater than 200 nm. 93.The method of claim 92, wherein the diameter is greater than 250 nm. 94.The method of claim 93, wherein the diameter is greater than 300 nm. 95.The composition of any of claims 56-94, wherein the aspect ratio of thesynthetic nanocarriers of the first population and/or second populationis greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7 or 1:10.
 96. Themethod of any of claims 56-95, wherein the composition further comprisesa pharmaceutically acceptable excipient.
 97. The method of any of claims56-96, wherein the method further comprises administering thetherapeutic protein to the subject.
 98. The method of claim 97, whereinthe therapeutic protein is administered prior to, concomitantly with orafter the administration of the composition.
 99. The method of any ofclaims 56-98, wherein one or more maintenance doses of the compositionis administered to the subject.
 100. The method of any of claims 56-99,wherein the method further comprises assessing the generation of anundesired immune response in the subject prior to and/or after theadministration of the composition and/or therapeutic protein.
 101. Themethod of claim 100, wherein the undesired immune response is thegeneration of therapeutic protein-specific antibodies and/or CD4+ T cellproliferation and/or activity and/or B cell proliferation and/oractivity.
 102. The method of any of 56-101, wherein the administering isby intravenous, intraperitoneal, transmucosal, oral, subcutaneous,pulmonary, intranasal, intradermal or intramuscular administration. 103.The method of claim 102, wherein the administering is by inhalation orintravenous, subcutaneous or transmucosal administration.
 104. A methodcomprising: (i) producing a first population of synthetic nanocarriersthat are coupled to immunosuppressants, and (ii) producing a secondpopulation of synthetic nanocarriers that are coupled to therapeuticprotein APC presentable antigens.
 105. The method of claim 104, whereinthe first population and second population are the same.
 106. The methodof claim 104 or 105, wherein the first and second populations ofsynthetic nanocarriers that are produced are as defined in any of claims1-38.
 107. The method of any of claims 104-106, wherein the methodfurther comprises producing a dosage form of a composition comprisingthe first and second populations of synthetic nanocarriers produced.108. The method of any of claims 104-107, wherein the method furthercomprises making a composition comprising the first population andsecond population of synthetic nanocarriers or the dosage form availableto a subject for administration.
 109. The method of any of claims104-108, wherein the method further comprises assessing the reduction ofan undesired immune response with a composition comprising the firstpopulation and second population of synthetic nanocarriers.
 110. Themethod of claim 109, wherein the undesired immune response is thegeneration of therapeutic protein-specific antibodies and/or CD4+ T cellproliferation and/or activity and/or B cell proliferation and/oractivity.
 111. A process for producing a composition or dosage formcomprising: (i) a first population of synthetic nanocarriers that arecoupled to immunosuppressants, and (ii) a second population of syntheticnanocarriers that are coupled to therapeutic protein APC presentableantigens, the process comprising the steps as defined in the method ofany of claims 104-110.
 112. A composition or dosage form obtainable bythe method or process of any one of claims 104-111.
 113. A compositionof any one of claims 1-38 and 112 or dosage form of claim 39 for use intherapy or prophylaxis.
 114. A composition of any one of claims 1-38 and112 or dosage form of claim 39 for use in a method of inducing atolerogenic immune response to therapeutic protein antigens, cell-basedtherapy, protein replacement therapy, protein supplementation therapy ora method as defined in any one of claims 40-110.
 115. Use of thecomposition of any one of claims 1-38 and 112 or dosage form of claim 39for the manufacture of a medicament for use in a method of inducing atolerogenic immune response to therapeutic protein antigens, cell-basedtherapy, protein replacement therapy, protein supplementation therapy ora method as defined in any one of claims 40-110.
 116. A dosage formcomprising the composition of any of claims 112-114.